Type II Interleukin-1 receptors

ABSTRACT

Type II IL-1 receptor (type II IL-1R) proteins, DNAs and expression vectors encoding type II IL-1R, and processes for producing type II IL-1R as products of recombinant cell culture, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. Ser. No. 08/091,519, filed Jul.12, 1993, issued as U.S. Pat. No. 5,350,683 on Sep. 27, 1994, nowallowed, which is a continuation of U.S. application Ser. No.07/701,415, filed May 16, 1991, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 07/627,071, filed Dec.13, 1990, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 07/573,576, filed Aug. 24, 1990, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 07/534,193,filed Jun. 5, 1990, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to cytokine receptors, and morespecifically, to Type II (B Cell) Interleukin-1 receptors.

Interleukin-1α (IL-1α) and Interleukin-1β (IL-1β) are distantly relatedpolypeptide hormones which play a central role in the regulation ofimmune and inflammatory responses. These two proteins act on a varietyof cell types and have multiple biological activities. The diversity ofbiological activity ascribed to IL-1α and IL-1β is mediated by specificplasma membrane receptors which bind both IL-1α and IL-1β. Due to thewide range of biological activities mediated by IL-1α and IL-1β it wasoriginally believed that the IL-1 receptors should be highly conservedin a variety of species and expressed on a large variety of cells.

Structural chracterization by ligand affinity cross-linking techniqueshas demonstrated that, despite their significant divergence in sequence,IL-1α and IL-1β bind to the same cell surface receptor molecule on Tcells and fibroblasts (Dower et al., Nature (London) 324:266, 1986; Birdet al., Nature (London) 324:263, 1986; Dower et al., Proc. Natl. Acad.Sci. USA 83:1060, 1986). The IL-1 receptor on murine and human T cellshas been identified by cDNA expression cloning and N-terminal sequenceanalysis as an integral membrane glycoprotein that binds IL-1α and IL-1βand has a molecular weight of 80,000 kDa (Sims et al., Science 241:585,1988; Sims et at., Proc. Natl. Acad. Sci. USA 6:8946, 1989).

Subsequent affinity cross-linking studies indicate that IL-1 receptorson the Epstein Barr virus (EBV)-transformed human B cell lines VDS-O and3B6, the EBV-positive Burkitt's lymphoma cell line Raji, and the murinepre-B cell line 70Z/3, have a molecular weight of 60,000 to 68,000 kDa(Matsushima et al., J. Immunol. 136:4496, 1986; Bensimon et al., J.Immunol. 142:2290, 1989; Bensimon et al., J. Immunol. 143:1168, 1989;Horuk et al., J. Biol. Chem. 262:16275, 1987; Chizzonite et at., Proc.Natl. Acad. Sci. USA 86:8029, 1989; Bomsztyk et al., Proc. Natl. Acad.Sci. USA 86:8034, 1989). Moreover, comparison of the biochemicalproperties and kinetic analysis of the IL- 1 receptor in the Raji B cellline with EL-4 murine T lymphoma cell line showed that Raji cells hadlower binding affinity but much higher receptor density per cell than asubclone of EL-4 T cells (Horuk et al., J. Biol. Chem. 262:16275, 1987).Raji cells also failed to internalize IL- 1 and demonstrated alteredreceptor binding affinities with IL-1 analogs. (Horuk et al., J. Biol.Chem. 262:16275, 1987). These data suggest that the IL-1 receptorsexpressed on B cells (referred to herein as type II IL-1 receptors) aredifferent from IL-1 receptors detected on T cells and other cell types(referred to herein as type I IL- 1 receptors).

In order to study the structural and biological characteristics of typeII IL-1R and the role played by type II IL-1R in the responses ofvarious cell populations to IL-1 stimulation, or to use type II IL-1Reffectively in therapy, diagnosis, or assay, homogeneous compositionsare needed. Such compositions are theoretically available viapurification of receptors expressed by cultured cells, or by cloning andexpression of genes encoding the receptors. Prior to the presentinvention, however, several obstacles prevented these goals from beingachieved.

First, no cell lines have previously been known to express high levelsof type II IL1R constitutively and continuously, and cell lines known toexpress type II IL-1R did so only in low numbers (500 to 2,000receptors/cell) which impeded efforts to purify receptors in amountssufficient for obtaining amino acid sequence information or generatingmonoclonal antibodies. The low numbers of receptors has also precludedany practical translation assay-based method of cloning.

Second, the significant differences in DNA sequence between type I IL-1Rand type II IL-1R has precluded cross-hybridization using a murine typeIL-1R cDNA (Bomsztyk et at., Proc. Natl. Acad. Sci. USA 86:8034, 1989,and Chizzonite et at., Proc. Natl. Acad. Sci. USA 86:8029, 1989).

Third, even if a protein composition of sufficient purity could beobtained to permit N-terminal protein sequencing, the degeneracy of thegenetic code may not permit one to define a suitable probe withoutconsiderable additional experimentation. Many iterative attempts may berequired to define a probe having the requisite specificity to identifya hybridizing sequence in a cDNA library. Although direct expressioncloning techniques avoid the need for repetitive screening usingdifferent probes of unknown specificity and have been useful in cloningother receptors (e.g., type I IL-1R), they are not sufficientlysensitive to be suitable for using in identifying type II IL-1R clonesfrom cDNA libraries derived from cells expressing low numbers of type IIIt,-1R.

Thus, efforts to purify the type II IL-1R or to clone or express genesencoding type II IL- 1R have been significantly impeded by lack ofpurified receptor, a suitable source of receptor mRNA, and by asufficiently sensitive cloning technique.

SUMMARY OF THE INVENTION

The present invention provides isolated type II IL-1R and isolated DNAsequences encoding type II IL-1R, in particular, human type II IL-1R, oranalogs thereof. Preferably, such DNA sequences are selected from thegroup consisting of (a) cDNA clones having a nucleotide sequence derivedfrom the coding region of a native type II IL-1R gene, such as clone 75;(b) DNA sequences capable of hybridization to the eDNA clones of (a)under moderately stringent conditions and which encode biologicallyactive IL-1R molecules; and (c) DNA sequences which are degenerate as aresult of the genetic code to the DNA sequences defined in (a) and (b)and which encode biologically active IL-1R molecules. The presentinvention also provides recombinant expression vectors comprising theDNA sequences defined above, recombinant type II IL-1R moleculesproduced using the recombinant expression vectors, and processes forproducing the recombinant type II IL1R molecules utilizing theexpression vectors.

The present invention also provides substantially homogeneous proteincompositions comprising type II IL-1R.

The present invention also provides compositions for use in therapy,diagnosis, assay of type II IL-1R, or in raising antibodies to type IIIL-1R, comprising effective quantities of soluble native or recombinantreceptor proteins prepared according to the foregoing processes.

These and other aspects of the present invention will become evidentupon reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scehmatic diagram of the expression plasmid pDC406. cDNAmolecules inserted at the Sal site are transcribed and translated usingregulatory elements derived from HIV and adenovirus. pDC406 containsorigins of replication derived from SV40, Epstein-Barr virus and pBR322.

FIG. 2 is a schematic diagram of the human and murine type II IL-1receptors and the various human and murine clones used to determine thesequences. Thin lines represent untranslated regions, while the codingregion is depicted by a box. The sections encoding the signal peptideare filled in; the transmembrane regions are cross-hatched; and thecytoplasmic portions are stippled. Potential N-linked glycosylationsites are marked by inverted triangles. The predictedimmunoglobulin-like disulfide bonds are also indicated by dashesconnecting two sulfide molecules (S-----S).

FIG. 3 compares the amino acid sequences of the human and murine type IIIL-1 receptors (as deduced from the cDNA clones) with the amino acidsequences of the human and murine type I IL-1 receptors (Sims et al.,Proc. Natl. Acad. Sci. USA 86:8946, 1989; Sims et at., Science 241:585,1988) and the amino acid sequences of the ST2 cellular gene (Tominaga,FEBS Lett. 258:301, 1989) and the B 15R open reading frame of vacciniavirus (Smith and Chan, J. Gen. Virology 72:511, 1991). Numbering beginswith the initiating methionine. The predicted position of the signalpeptide cleavage in each sequences was determined according to themethod described by von Heijne, Nucl. Acids. Res. 14:4683, 1986, and isindicated by a gap between the putative signal peptide and the main bodyof the protein. The predicated transmembrane and cytoplasmic regions forthe type II IL-1 receptors are shown on the bottom line, and areseparated from one another by a gap. Residues conserved in all four IL-1receptor sequences are presented in white on a black background.Residues conserved in type II receptors that are also found in one ofthe other sequences are shaded; residues conserved in type I IL-1receptors that are found in one of the other sequences are boxed.Cysteine residues involved in forming the disulfide bonds characteristicof the immunolgobulin fold are marked with solid dots, while the extratwo pairs of cysteines found in the type I IL-1 receptor and in some ofthe other sequences are indicated by stars. The approximate boundariesof domains 1, 2 and 3 are indicated above the lines. The predictedsignal peptide cleavage in the type II IL-1 receptors follow Ala13,resulting in an unusually short signal peptide and an N-terminalextension of 12 (human) or 23 (mouse) amino acids beyond the pointcorresponding to the mature N-terminus of the human or mouse type I IL-1receptor. Other less favored but still acceptable sites of cleavage inthe murine type II IL-1 receptor are after Thr15 or Pro 17. Thissequence alignment was made by hand and does not represent anobjectively optimized alignment of the sequences. The nucleotide andamino acid sequences of the full length and soluble human and murinetype H IL-1 receptor cDNAs are also set forth in the Sequence Listingherein.

FIG. 4 shows an autoradiograph of an SDS/PAGE gel with crosslinked IL-1receptors. Cells expressing IL-1 receptors were cross-linked to ¹²⁵I-IL-1 in the absence or presence of the cognate unlabeled IL-1competitor, extracted, electrophoresed and autoradiographed as describedin Example 6. Recombinant receptors were expressed transiently inCV1/EBNA cells. The cell lines used for cross-linking to naturalreceptors were KB (ATCC CCL 1717) (for human type I IL-1R), CB23 (forhuman type II IL-1R), EL4 (ATCC TIB 39) (for murine type I IL-1R), and70Z/3 (ATCC TIB 158) (for murine type II IL- 1R ).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"IL-1" refers collectively to IL-1 α and IL-1β.

"Type II Interleukin-1 receptor" and "type II IL-1R" refer to proteinswhich are capable of binding Interleukin-1 (IL-1) molecules. The maturefull-length human type II IL-1R is a glycoprotein having an apparentmolecular weight of approximately 60-68 kDa. Specific examples of typeII IL-1R proteins are shown in SEQ ID NO: 1 and SEQ ID NO: 12. As usedherein, the above terms include analogs or subunits of native type IIIL-1R proteins with IL-1-binding activity. Specifically included aretruncated or soluble forms of type II IL-1R protein, as defined below.In the absence of any species designation, type II IL-1R refersgenerically to mammalian type II IL-1R, which includes, but is notlimited to, human, murine, and bovine type II IL-1R. Similarly, in theabsence of any specific designation for deletion mutants, the term typeII IL-1R means all forms of type II IL-1 R, including mutants andanalogs which possess type II IL-1R biological activity. " Interleukin-1Receptor" or "IL-1R" refers collectively to type I IL-1 receptor andtype II IL-1 receptor.

"Soluble type II IL-1R" as used in the context of the present inventionrefer to proteins, or substantially equivalent analogs, which aresubstantially similar to all or part of the extracellular region of anative type II IL-1R, and are secreted by the cell but retain theability to bind IL-1 or inhibit IL-1 signal transduction activity viacell surface bound IL-1R proteins. Soluble type II IL-1R proteins mayalso include part of the transmembrane region, provided that the solubletype II IL-1R protein is capable of being secreted from the cell.Specific examples of soluble type II IL-1R proteins include proteinshaving the sequence of amino acids 1-330 or amino acids 1-333 of SEQ IDNO: 1 and amino acids 1-342 and amino acids 1-345 of SEQ ID NO:12.Inhibition of IL-1 signal transduction activity can be determined usingprimary cells or cells lines which express an endogenous IL-1R and whichare biologically responsive to IL- 1 or which, when transfected withrecombinant IL-1R DNAs, are biologically responsive to IL-1. The cellsare then contacted with IL-1 and the resulting metabolic effectsexamined. If an effect results which is attributable to the action ofthe ligand, then the recombinant receptor has signal transductionactivity. Exemplary procedures for determining whether a polypeptide hassignal transduction activity are disclosed by Idzerda et al., J. Exp.Med. 171:861 (1990); Curtis et al., Proc. Natl. Acad. Sci. USA 86:3045(1989); Prywes et al., EMBO J. 5:2179 (1986) and Chou et al., J. Biol.Chem. 262:1842 (1987).

The term "isolated" or "purified", as used in the context of thisspecification to define the purity of type II IL-1R protein or proteincompositions, means that the protein or protein composition issubstantially free of other proteins of natural or endogenous origin andcontains less than about 1% by mass of protein contaminants residual ofproduction processes. Such compositions, however, can contain otherproteins added as stabilizers, carriers, excipients or co-therapeutics.Type II IL-1R is "isolated" if it is detectable as a single protein bandin a polyacrylamide gel by silver staining.

The term "substantially similar," when used to define either amino acidor nucleic acid sequences, means that a particular subject sequence, forexample, a mutant sequence, varies from a reference sequence by one ormore substitutions, deletions, or additions, the net effect of which isto retain biological activity of the type II IL-1R protein as may bedetermined, for example, in a type II IL-1R binding assays, such as isdescribed in Example 5 below. Alternatively, nucleic acid subunits andanalogs are "substantially similar" to the specific DNA sequencesdisclosed herein if: (a) the DNA sequence is derived from the codingregion of SEQ ID NO:1 or SEQ ID NO:12; (b) the DNA sequence is capableof hybridization to DNA sequences of (a) under moderately stringentconditions (25% formamide, 42° C., 2×SSC) or alternatively under morestringent conditions (50% formamide, 50° C., 2×SSC or 50% formamide, 42°C., 2×SSC) and which encode biologically active IL-1R molecules; or DNAsequences which are degenerate as a result of the genetic code to theDNA sequences defined in (a) or (b) and which encode biologically activeIL- 1R molecules.

"Recombinant," as used herein, means that a protein is derived fromrecombinant (e.g., microbial or mammalian) expression systems."Microbial" refers to recombinant proteins made in bacterial or fungal(e.g., yeast) expression systems. As a product, "recombinant microbial"defines a protein essentially free of native endogenous substances andunaccompanied by associated native glycosylation. Protein expressed inmost bacterial cultures, e.g., E. coli, will be free of glycan; proteinexpressed in yeast may have a glycosylation pattern different from thatexpressed in mammalian cells.

"Biologically active," as used throughout the specification as acharacteristic of type II IL-1R, means either that a particular moleculeshares sufficient amino acid sequence similarity with SEQ ID NO:2 or SEQID NO:13 to be capable of binding detectable quantities of IL-1,preferably at least 0.01 nmoles IL-1 per nanomole type II IL-1R, forexample, as a component of a hybrid receptor construct. More preferably,biologically active type II IL-1R within the scope of the presentinvention is capable of binding greater than 0.1 nanomoles IL-1 pernanomole receptor, and most preferably, greater than 0.5 nanomoles IL-1per nanomole receptor.

"DNA sequence" refers to a DNA polymer, in the form of a separatefragment or as a component of a larger DNA construct, which has beenderived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesequence and its component nucleotide sequences by standard biochemicalmethods, for example, using a cloning vector. Such sequences arepreferably provided in the form of an open reading frame uninterruptedby internal nontranslated sequences, or introns, which are typicallypresent in eukaryotic genes. However, it will be evident that genomicDNA containing the relevant sequences could also be used. Sequences ofnon-translated DNA may be present 5' or 3' from the open reading frame,where the same do not interfere with manipulation or expression of thecoding regions.

"Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.DNA sequences encoding the proteins provided by this invention areassembled from cDNA fragments and short oligonucleotide linkers, or froma series of oligonucleotides, to provide a synthetic gene which iscapable of being expressed in a recombinant transcriptional unit.

"Recombinant expression vector" refers to a plasmid comprising atranscriptional unit comprising an assembly of (1) a genetic element orelements having a regulatory role in gene expression, for example,promoters or enhancers, (2) a structural or coding sequence which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription and translation initiation and termination sequences.Structural elements intended for use in yeast expression systemspreferably include a leader sequence enabling extracellular secretion oftranslated protein by a host cell. Alternatively, where recombinantprotein is expressed without a leader or transport sequence, it mayinclude an N-terminal methionine residue. This residue may optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

"Recombinant microbial expression system" means a substantiallyhomogeneous monoculture of suitable host microorganisms, for example,bacteria such as E. coli or yeast such as S. cerevisiae, which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit as a component of aresident plasmid. Generally, cells constituting the system are theprogeny of a single ancestral transformant. Recombinant expressionsystems as defined herein will express heterologous protein uponinduction of the regulatory elements linked to the DNA sequence orsynthetic gene to be expressed.

Isolation of cDNAs Encoding Type H IL-1R

In order to secure a human coding sequence, a DNA sequence encodinghuman type II IL-1R (see SEQ ID NO:1) was isolated from a cDNA libraryprepared using standard methods by reverse transcription ofpolyadenylated RNA isolated from the human B cell lymphoblastoid lineCB23, described by Benjamin & Dower, Blood 75:2017, 1990. Briefly, theCB23 cell line is an EBV-transformed cord blood (CB) lymphocyte cellline, which was derived using the methods described by Benjamin et al.,Proc. Natl. Acad. Sci. USA 81:3547, 1984.

The CB23 library was screened by modified direct expression of pooledcDNA fragments in the monkey kidney cell line CV-1/EBNA-1 using amammalian expression vector (pDC406) that includes regulatory sequencesderived from SV40 and human immunodeficiency virus (HIV), andEpstein-Barr virus (EBV). The CV-1/EBNA-1 cell line was derived bytransfection of the CV-1 cell line with the gene encoding Epstein-Barrvirus nuclear antigen-1 (EBNA-1) and constitutively expresses EBNA-1driven from the human CMV immediate-early enhancer/promoter. The EBNA-1gene allows the episomal replication of expression vectors such aspDC406 that contain the EBV origin of replication.

Transfectants expressing biologically active type II IL-1R wereinitially identified using a modified slide autoradiographic technique,substantially as described by Gearing et al., EMBO J. 8:3667, 1989.Briefly, CV-1/EBNA-1 cells were transfected with miniprep DNA in pDC406from pools of cDNA clones directly on glass slides and cultured for 2-3days to permit transient expression of type II IL-1R. The slidescontaining the transfected cells were then incubated with mediumcontaining ¹²⁵ I-IL-1β, washed to remove unbound labeled IL-1β, fixedwith glutaraldehyde, and dipped in liquid photographic emulsion andexposed in the dark. After developing the slides, they were individuallyexamined with a microscope and positive cells expressing type II IL-1Rwere identified by the presence of autoradiographic silver grainsagainst a light background.

Using this approach, approximately 250,000 cDNAs were screened in poolsof approximately 3,000 cDNAs using the slide autoradiographic methoduntil assay of one transfectant pool showed multiple cells clearlypositive for IL-1β binding. This pool was then partitioned into pools of500 and again screened by slide autoradiography and a positive pool wasidentified. This pool was further partitioned into pools of 75 andscreened by plate binding assays analyzed by quantitation of bound ¹²⁵I-IL-1β. The cells were scraped off and counted to determine which poolof 75 was positive. Individual colonies from this pool of 75 werescreened until a single clone (clone 75) was identified which directedsynthesis of a surface protein with detectable IL-1β binding activity.This clone was isolated, and its insert was sequenced to determine thesequence of the human type II IL-1R cDNA clone 75 (SEQ ID NO:1). ThepDC406 cloning vector containing the human type II IL-1R cDNA,designated pHuTYPE II IL-1R 75, was deposited with the American TypeCulture Collection, Rockville, Md., USA (ATCC) on Jun. 5, 1990 underaccession number 68337. The deposit was made under the conditions of theBudapest Treaty.

A probe may be constructed from the human sequence and used to screenvarious other mammalian cDNA libraries. cDNA clones which hybridized tothe human probe are then isolated and sequenced.

Like most mammalian genes, mammalian type II IL-1R is presumably encodedby multi-exon genes. Alternative mRNA constructs which can be attributedto different mRNA splicing events following transcription, and whichshare large regions of identity or similarity with the cDNAs claimedherein, are considered to be within the scope of the present invention.

Proteins and Analogs

The present invention provides isolated recombinant mammalian type IIIL-1R polypeptides. Isolated type II IL-1R polypeptides of thisinvention are substantially free of other contaminating materials ofnatural or endogenous origin and contain less than about 1% by mass ofprotein contaminants residual of production processes. The native humantype II IL-1R molecules are recovered from cell lysates as glycoproteinshaving an apparent molecular weight by SDS-PAGE of about 60-68kilodaltons (kDa). The type II IL-1R polypeptides of this invention areoptionally without associated native-pattern glycosylation.

Mammalian type II IL-1R of the present invention includes, by way ofexample, primate, human, murine, canine, feline, bovine, ovine, equine,caprine and porcine type II IL-1R. Mammalian type II IL-1R can beobtained by cross species hybridization, using a single stranded cDNAderived from the human type II IL-1R DNA sequence, for example, clone75, as a hybridization probe to isolate type II IL-1R cDNAs frommammalian cDNA libraries. DNA sequences which encode IL-IR-II, possiblyin the form of alternate splicing arrangements, can be isolated from thefollowing cells and tissues: B lymphoblastoid lines (such as CB23, CB33,Raji, RPMI1788, ARH77), resting and especially activated peripheralblood T cells, monocytes, the monocytic cell line THP1, neutrophils,bone marrow, placenta, endothelial cells, keratinocytes (especiallyactivated), and HepG2 cells.

Derivatives of type II IL-1R within the scope of the invention alsoinclude various structural forms of the primary protein which retainbiological activity. Due to the presence of ionizable amino and carboxylgroups, for example, a type II IL-1R protein may be in the form ofacidic or basic salts, or may be in neutral form. Individual amino acidresidues may also be modified by oxidation or reduction.

The primary amino acid structure may be modified by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like, or by creatingamino acid sequence mutants. Covalent derivatives are prepared bylinking particular functional groups to type II IL-1R amino acid sidechains or at the N- or C-termini. Other derivatives of type II IL-1Rwithin the scope of this invention include covalent or aggregativeconjugates of type II IL-1R or its fragments with other proteins orpolypeptides, such as by synthesis in recombinant culture as N-terminalor C-terminal fusions. For example, the conjugated peptide may be a asignal (or leader) polypeptide sequence at the N-terminal region of theprotein which cotranslationally or post-translationally directs transferof the protein from its site of synthesis to its site of function insideor outside of the cell membrane or wall (e.g., the yeast α-factorleader). Type II IL-1R protein fusions can comprise peptides added tofacilitate purification or identification of Type II IL-1R (e.g.,poly-His). The amino acid sequence of type II IL-1R can also be linkedto the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (Hopp et al.,Bio/Technology 6:1204,1988.) The latter sequence is highly antigenic andprovides an epitope reversibly bound by a specific monoclonal antibody,enabling rapid assay and facile purification of expressed recombinantprotein. This sequence is also specifically cleaved by bovine mucosalenterokinase at the residue immediately following the Asp-Lys pairing.Fusion proteins capped with this peptide may also be resistant tointracellular degradation in E. coli.

Type II IL-1R derivatives may also be used as immunogens, reagents inreceptor-based immunoassays, or as binding agents for affinitypurification procedures of IL-1 or other binding ligands. type II IL-1Rderivatives may also be obtained by cross-linking agents, such asM-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, atcysteine and lysine residues. Type II IL-1R proteins may also becovalently bound through reactive side groups to various insolublesubstrates, such as cyanogen bromide-activated, bisoxirane-activated,carbonyldiimidazole-activated or tosyl-activated agarose structures, orby adsorbing to polyolefin surfaces (with or without glutaraldehydecross-linking). Once bound to a substrate, type II IL-1R may be used toselectively bind (for purposes of assay or purification) anti-type IIIL-1R antibodies or IL-1.

The present invention also includes type II IL-1R with or withoutassociated native-pattern glycosylation. Type II IL-1R expressed inyeast or mammalian expression systems, e.g., COS-7 cells, may be similaror slightly different in molecular weight and glycosylation pattern thanthe native molecules, depending upon the expression system. Expressionof type II IL-1R DNAs in bacteria such as E. coil providesnon-glycosylated molecules. Functional mutant analogs of mammalian typeII IL-1R having inactivated N-glycosylation sites can be produced byoligonucleotide synthesis and ligation or by site-specific mutagenesistechniques. These analog proteins can be produced in a homogeneous,reduced-carbohydrate form in good yield using yeast expression systems.N-glycosylation sites in eukaryotic proteins are characterized by theamino acid triplet Asn-A₁ -Z, where A₁ is any amino acid except Pro, andZ is Ser or Thr. In this sequence, asparagine provides a side chainamino group for covalent attachment of carbohydrate. Examples ofN-glycosylation sites in human type II IL-1R are amino acids 66-68,72-74, 112-114, 219-221, and 277-279 in SEQ ID NO:1. Such sites can beeliminated by substituting another amino acid for Asn or for residue Z,deleting Asn or Z, or inserting a non-Z amino acid between A₁ and Z, oran amino acid other than Asn between Asn and A₁.

Type II IL-1R derivatives may also be obtained by mutations of type IIIL-1R or its subunits. A type II IL-1R mutant, as referred to herein, isa polypeptide homologous to type II IL-1R but which has an amino acidsequence different from native type II IL-1R because of a deletion,insertion or substitution.

Bioequivalent analogs of type II IL-1R proteins may be constructed by,for example, making various substitutions of residues or sequences ordeleting terminal or internal residues or sequences not needed forbiological activity. For example, cysteine residues can be deleted orreplaced with other amino acids to prevent formation of unnecessary orincorrect intramolecular disulfide bridges upon renaturation. Otherapproaches to mutagenesis involve modification of adjacent dibasic aminoacid residues to enhance expression in yeast systems in which KEX2protease activity is present. Generally, substitutions should be madeconservatively; i.e., the most preferred substitute amino acids arethose having physiochemical characteristics resembling those of theresidue to be replaced. Similarly, when a deletion or insertion strategyis adopted, the potential effect of the deletion or insertion onbiological activity should be considered. Substantially similarpolypeptide sequences, as defined above, generally comprise a likenumber of amino acids sequences, although C-terminal truncations for thepurpose of constructing soluble type II IL-1Rs will contain fewer aminoacid sequences. In order to preserve the biological activity of type IIIL-1Rs, deletions and substitutions will preferably result in homologousor conservatively substituted sequences, meaning that a given residue isreplaced by a biologically similar residue. Examples of conservativesubstitutions include substitution of one aliphatic residue for another,such as Ile, Val, Leu, or Ala for one another, or substitutions of onepolar residue for another, such as between Lys and Arg; Glu and Asp; orGln and Asn. Other such conservative substitutions, for example,substitutions of entire regions having similar hydrophobicitycharacteristics, are well known. Moreover, particular amino aciddifferences between human, murine and other mammalian type II IL-1Rs issuggestive of additional conservative substitutions that may be madewithout altering the essential biological characteristics of type IIIL-1R.

Subunits of type II IL-1R may be constructed by deleting terminal orinternal residues or sequences. The present invention contemplates, forexample, C terminal deletions which result in soluble type II IL-1Rconstructs corresponding to all or part of the extracellular region oftype II IL-1R. The resulting protein preferably retains its ability tobind IL-1. Particularly preferred sequences include those in which thetransmembrane region and intracellular domain of type II IL-1R aredeleted or substituted with hydrophilic residues to facilitate secretionof the receptor into the cell culture medium. Soluble type II IL-1Rproteins may also include part of the transmembrane region, providedthat the soluble type II IL-1R protein is capable of being secreted fromthe cell. For example, soluble human type II IL-1R may comprise thesequence of amino acids 1-333 or amino acids 1-330 of SEQ ID NO:1 andamino acids 1-345 and amino acids 1-342 of SEQ ID NO:12. Alternatively,soluble type II IL-1R proteins may be derived by deleting the C-terminalregion of a type II IL-1R within the extracellular region which are notnecessary for IL-1 binding. For example, C-terminal deletions may bemade to proteins having the sequence of SEQ D NO:1 and SEQ D NO:12following amino acids 313 and 325, respectively. These amino acids arecysteines which are believed to be necessary to maintain the tertiarystructure of the type II IL-1R molecule and permit binding of the type HIL-1R molecule to IL-1. Soluble type II IL-1R constructs are constructedby deleting the 3'-terminal region of a DNA encoding the type II IL-1Rand then inserting and expressing the DNA in appropriate expressionvectors. Exemplary methods of constructing such soluble proteins aredescribed in Examples 2 and 4. The resulting soluble type II IL-1Rproteins are then assayed for the ability to bind IL-1, as described inExample 5. Both the DNA sequences encoding such soluble type II IL-1Rsand the biologically active soluble type II IL-1R proteins resultingfrom such constructions are contemplated to be within the scope of thepresent invention.

Mutations in nucleotide sequences constructed for expression of analogtype II IL-1R must, of course, preserve the reading frame phase of thecoding sequences and preferably will not create complementary regionsthat could hybridize to produce secondary mRNA structures such as loopsor hairpins which would adversely affect translation of the receptormRNA. Although a mutation site may be predetermined, it is not necessarythat the nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed type II IL-1R mutants screened for the desired activity.

Not all mutations in the nucleotide sequence which encodes type II IL-1Rwill be expressed in the final product, for example, nucleotidesubstitutions may be made to enhance expression, primarily to avoidsecondary structure loops in the transcribed mRNA (see EPA 75,444A,incorporated herein by reference), or to provide codons that are morereadily translated by the selected host, e.g., the well-known E. colipreference codons for E. coli expression.

Mutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, Jan. 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981); andU.S. Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, andare incorporated by reference herein.

Both monovalent forms and polyvalent forms of type II IL-1R are usefulin the compositions and methods of this invention. Polyvalent formspossess multiple type II IL1R binding sites for IL-1 ligand. Forexample, a bivalent soluble type II IL-1R may consist of two tandemrepeats of the extracellular region of type II IL-1R, separated by alinker region. Alternate polyvalent forms may also be constructed, forexample, by chemically coupling type II IL-1R to any clinicallyacceptable carrier molecule, a polymer selected from the groupconsisting of Ficoll, polyethylene glycol or dextran using conventionalcoupling techniques. Alternatively, type II IL-1R may be chemicallycoupled to biotin, and the biotin-type H IL- 1R conjugate then allowedto bind to avidin, resulting in tetravalent avidin/biotin/type II IL-1Rmolecules. Type II IL-1R may also be covalently coupled to dinitrophenol(DNP) or trinitrophenol (TNP) and the resulting conjugate precipitatedwith anti-DNP or anti-TNP-IgM, to form decameric conjugates with avalency of 10 for type II IL-1R binding sites.

A recombinant chimeric antibody molecule may also be produced havingtype II IL1R sequences substituted for the variable domains of either orboth of the immunoglubulin molecule heavy and light chains and havingunmodified constant region domains. For example, chimeric type IIIL-1R/IgG₁ may be produced from two chimeric genes--a type IIIL-1R/human κ light chain chimera (type II IL-1R/C.sub.κ) and a type IIIL-1R/human γ1 heavy chain chimera (type II IL-1R/C.sub.γ-1). Followingtranscription and translation of the two chimeric genes, the geneproducts assemble into a single chimeric antibody molecule having typeII IL-1R displayed bivalently. Such polyvalent forms of type II IL-1Rmay have enhanced binding affinity for IL-1 ligand. Additional detailsrelating to the construction of such chimeric antibody molecules aredisclosed in WO 89/09622 and EP 315062.

Expression of Recombinant Type II IL-1R

The present invention provides recombinant expression vectors to amplifyor express DNA encoding type II IL-1R. Recombinant expression vectorsare replicable DNA constructs which have synthetic or cDNA-derived DNAfragments encoding mammalian type II IL-1R or bioequivalent analogsoperably linked to suitable transcriptional or translational regulatoryelements derived from mammalian, microbial, vital or insect genes. Atranscriptional unit generally comprises an assembly of (1) a geneticelement or elements having a regulatory role in gene expression, forexample, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences, as described in detail below. Such regulatoryelements may include an operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding sims. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants mayadditionally be incorporated. DNA regions are operably linked when theyare functionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operably linked to DNA for a polypeptideif it is expressed as a precursor which participates in the secretion ofthe polypeptide; a promoter is operably linked to a coding sequence ifit controls the transcription of the sequence; or a ribosome bindingsite is operably linked to a coding sequence if it is positioned so asto permit translation. Generally, operably linked means contiguous and,in the case of secretory leaders, contiguous and in reading frame.Structural elements intended for use in yeast expression systemspreferably include a leader sequence enabling extracellular secretion oftranslated protein by a host cell. Alternatively, where recombinantprotein is expressed without a leader or transport sequence, it mayinclude an N-terminal methionine residue. This residue may optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

DNA sequences encoding mammalian type II IL-1Rs which are to beexpressed in a microorganism will preferably contain no introns thatcould prematurely terminate transcription of DNA into mRNA; however,premature termination of transcription may be desirable, for example,where it would result in mutants having advantageous C-terminaltruncations, for example, deletion of a transmembrane region to yield asoluble receptor not bound to the cell membrane. Due to code degeneracy,there can be considerable variation in nucleotide sequences encoding thesame amino acid sequence. Other embodiments include sequences capable ofhybridizing to clone 75 under moderately stringent conditions (50° C.,2×SSC) and other sequences hybridizing or degenerate to those whichencode biologically active type II IL-1R polypeptides.

Recombinant type II IL-1R DNA is expressed or amplified in a recombinantexpression system comprising a substantially homogeneous monoculture ofsuitable host microorganisms, for example, bacteria such as E. coli oryeast such as S. cerevisiae, which have stably integrated (bytransformation or transfection) a recombinant transcriptional unit intochromosomal DNA or carry the recombinant transcriptional unit as acomponent of a resident plasmid. Generally, cells constituting thesystem are the progeny of a single ancestral transformant. Recombinantexpression systems as defined herein will express heterologous proteinupon induction of the regulatory elements linked to the DNA sequence orsynthetic gene to be expressed.

Transformed host cells are cells which have been transformed ortransfected with type II IL-1R vectors constructed using recombinant DNAtechniques. Transformed host cells ordinarily express type II IL-1R, buthost cells transformed for purposes of cloning or amplifying type HIL-1R DNA do not need to express type II IL-1R. Expressed type II IL-1Rwill be deposited in the cell membrane or secreted into the culturesupernatant, depending on the type II IL-1R DNA selected. Suitable hostcells for expression of mammalian type II IL-1R include prokaryotes,yeast or higher eukaryotic cells under the control of appropriatepromoters. Prokaryotes include gram negative or gram positive organisms,for example E. coli or bacilli. Higher eukaryotic cells includeestablished cell lines of mammalian origin as described below. Cell-freetranslation systems could also be employed to produce mammalian type IIIL-1R using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, and mammalian cellular hosts are described byPouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.,1985), the relevant disclosure of which is hereby incorporated byreference.

Prokaryotic expression hosts may be used for expression of type II IL-1Rthat do not require extensive proteolytic and disulfide processing.Prokaryotic expression vectors generally comprise one or more phenotypicselectable markers, for example a gene encoding proteins conferringantibiotic resistance or supplying an autotrophic requirement, and anorigin of replication recognized by the host to ensure amplificationwithin the host. Suitable prokaryotic hosts for transformation includeE. coli, Bacillus subtilis, Salmonella typhimurium, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphyolococcus,although others may also be employed as a matter of choice.

Useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1(Promega Biotec, Madison, Wis., USA). These pBR322 "backbone" sectionsare combined with an appropriate promoter and the structural sequence tobe expressed. E. coli is typically transformed using derivatives ofpBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene2:95, 1977). pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides simple means for identifying transformedcells.

Promoters commonly used in recombinant microbial expression vectorsinclude the β-lactamase (penicillinase) and lactose promoter system(Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544,1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, 1980; and EPA 36,776) and tac promoter (Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412,1982). A particularly useful bacterial expression system employs thephage λ, P_(L) promoter and cI857ts thermolabile repressor. Plasmidvectors available from the American Type Culture Collection whichincorporate derivatives of the λ P_(L) promoter include plasmid pHUB2,resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E.coli RR1 (ATCC 53082).

Recombinant type II IL-1R proteins may also be expressed in yeast hosts,preferably from the Saccharornyces species, such as S. cerevisiae. Yeastof other genera, such as Pichia or Kluyveromyces may also be employed.Yeast vectors will generally contain an origin of replication from the2μ yeast plasmid or an autonomously replicating sequence (ARS),promoter, DNA encoding type II IL-1 R, sequences for polyadenylation andtranscription termination and a selection gene. Preferably, yeastvectors will include an origin of replication and selectable markerpermitting transformation of both yeast and E. coli, e.g., theampicillin resistance gene of E. coli and S. cerevisiae TRP1 or URA3gene, which provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, and a promoter derived from ahighly expressed yeast gene to induce transcription of a structuralsequence downstream. The presence of the TRP1 or URA3 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan oruracil.

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pUC18for selection and replication in E. coli (Amp^(r) gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and a-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). The yeast α-factor leader, which directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed. See, e.g., Kurjan et al., Cell30:933, 1982; and Bitter et at., Proc. Natl. Acad. Sci. USA 81:5330,1984. The leader sequence may be modified to contain, near its 3' end,one or more useful restriction sites to facilitate fusion of the leadersequence to foreign genes.

Suitable yeast transformation protocols are known to those of skill inthe art; an exemplary technique is described by Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp⁺ transformants ina selective medium consisting of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil or URA+tranformants in medium consisting of 0.67% YNB, with amino acids andbases as described by Sherman et al., Laboratory Course Manual forMethods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1986.

Host strains transformed by vectors comprising the ADH2 promoter may begrown for expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% or 4% glucose supplemented with 80 μg/ml adeninc and 80μg/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustionof medium glucose. Crude yeast supernatants are harvested by filtrationand held at 4° C. prior to further purification.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells is particularly preferred because suchproteins are generally correctly folded, appropriately modified andcompletely functional. Examples of suitable mammalian host cell linesinclude the COS-7 lines of monkey kidney cells, described by Gluzman(Cell 23:175, 1981), and other cell lines capable of expressing anappropriate vector including, for example, L cells, C127, 3T3, Chinesehamster ovary (CHO), HeLa and BHK cell lines. Mammalian expressionvectors may comprise nontranscribed elements such as an origin ofreplication, a suitable promoter and enhancer linked to the gene to beexpressed, and other 5' or 3' flanking nontranscribed sequences, and 5'or 3' nontranslated sequences, such as necessary ribosome binding sites,a polyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988).

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers et al., Nature 273:113, 1978). Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind 3 site toward the Bgl1 site located inthe viral origin of replication is included. Further, mammalian genomictype II IL-1R promoter, control and/or signal sequences may be utilized,provided such control sequences are compatible with the host cellchosen. Additional details regarding the use of a mammalian highexpression vector to produce a recombinant mammalian type II IL-1R areprovided in Examples 2 below. Exemplary vectors can be constructed asdisclosed by Okayama and Berg (Mol. Cell. Biol. 280, 1983).

A useful system for stable high level expression of mammalian receptorcDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986).

In preferred aspects of the present invention, recombinant expressionvectors comprising type II IL-1R cDNAs are stably integrated into a hostcell's DNA. Elevated levels of expression product is achieved byselecting for cell lines having amplified numbers of vector DNA. Celllines having amplified numbers of vector DNA are selected, for example,by transforming a host cell with a vector comprising a DNA sequencewhich encodes an enzyme which is inhibited by a known drug. The vectormay also comprise a DNA sequence which encodes a desired protein.Alternatively, the host cell may be co-transformed with a second vectorwhich comprises the DNA sequence which encodes the desired protein. Thetransformed or co-transformed host cells are then cultured in increasingconcentrations of the known drug, thereby selecting for drug-resistantcells. Such drug-resistant cells survive in increased concentrations ofthe toxic drug by overproduction of the enzyme which is inhibited by thedrug, frequently as a result of amplification of the gene encoding theenzyme. Where drug resistance is caused by an increase in the copynumber of the vector DNA encoding the inhibitable enzyme, there is aconcomitant co-amplification of the vector DNA encoding the desiredprotein (e.g., type II IL-1R) in the host cell's DNA.

A preferred system for such co-amplification uses the gene fordihydrofolate reductase (DHFR), which can be inhibited by the drugmethotrexate (MTX). To achieve co-amplification, a host cell which lacksan active gene encoding DHFR is either transformed with a vector whichcomprises DNA sequence encoding DHFR and a desired protein, or isco-transformed with a vector comprising a DNA sequence encoding DHFR anda vector comprising a DNA sequence encoding the desired protein. Thetransformed or co-transformed host cells are cultured in mediacontaining increasing levels of MTX, and those cells lines which surviveare selected.

A particularly preferred co-amplification system uses the gene forglutamine synthetase (GS), which is responsible for the synthesis ofglutamine from glutamate and ammonia using the hydrolysis of ATP to ADPand phosphate to drive the reaction. GS is subject to inhibition by avariety of inhibitors, for example methionine sulphoximine (MSX). Thus,type II IL-1R can be expressed in high concentrations by co-amplifyingcells transformed with a vector comprising the DNA sequence for GS and adesired protein, or co-transformed with a vector comprising a DNAsequence encoding GS and a vector comprising a DNA sequence encoding thedesired protein, culturing the host cells in media containing increasinglevels of MSX and selecting for surviving cells. The GS co-amplificationsystem, appropriate recombinant expression vectors and cells lines, aredescribed in the following PCT applications: WO 87/04462, WO 89/01036,WO 89/10404 and WO 86/05807.

Recombinant proteins are preferably expressed by co-amplification ofDHFR or GS in a mammalian host cell, such as Chinese Hamster Ovary (CHO)cells, or alternatively in a murine myeloma cell line, such asSP2/0-Ag14 or NS0 or a rat myeloma cell line, such as YB2/3.0-Ag20,disclosed in PCT applications WO/89/10404 and WO 86/05807.

A preferred eukaryotic vector for expression of type II IL-1R DNA isdisclosed below in Example 2. This vector, referred to as pDC406, wasderived from the mammalian high expression vector pDC201 and containsregulatory sequences from SV40, HIV and EBV.

Purification of Recombinant Type II IL-1R

Purified mammalian type II IL-1Rs or analogs are prepared by culturingsuitable host/vector systems to express the recombinant translationproducts of the DNAs of the present invention, which are then purifiedfrom culture media or cell extracts.

For example, supernatants from systems which secrete recombinant solubletype II IL-1R protein into culture media can be first concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a suitablepurification matrix. For example, a suitable affinity matrix cancomprise an IL-1 or lectin or antibody molecule bound to a suitablesupport. Alternatively, an anion exchange resin can be employed, forexample, a matrix or substrate having pendant diethylaminoethyl (DEAE)groups. The matrices can be acrylamide, agarose, dextran, cellulose orother types commonly employed in protein purification. Alternatively, acation exchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are preferred.

Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a type II IL-1R composition. Some or all ofthe foregoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of recombinant mammalian type II IL-1R can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express soluble mammalian type II IL-1R as asecreted protein greatly simplifies purification. Secreted recombinantprotein resulting from a large-scale fermentation can be purified bymethods analogous to those disclosed by Urdal et al. (J. Chromatog.296:171, 1984). This reference describes two sequential, reversed-phaseHPLC steps for purification of recombinant human GM-CSF on a preparativeHPLC column.

Human type II IL-1R synthesized in recombinant culture is characterizedby the presence of non-human cell components, including proteins, inamounts and of a character which depend upon the purification stepstaken to recover human type II IL-1R from the culture. These componentsordinarily will be of yeast, prokaryotic or non-human higher eukaryoticorigin and preferably are present in innocuous contaminant quantities,on the order of less than about 1 percent by weight. Further,recombinant cell culture enables the production of type II IL-1R free ofproteins which may be normally associated with type II IL-1R as it isfound in nature in its species of origin, e.g. in cells, cell exudatesor body fluids.

Therapeutic Administration of Recombinant Soluble Type H IL-1R

The present invention provides methods of using therapeutic compositionscomprising an effective amount of soluble type II IL-1R proteins and asuitable diluent and carrier, and methods for suppressing IL-1-dependentimmune responses in humans comprising administering an effective amountof soluble type II IL-1R protein.

For therapeutic use, purified soluble type II IL-1R protein isadministered to a patient, preferably a human, for treatment in a mannerappropriate to the indication. Thus, for example, soluble type II IL-1Rprotein compositions can be administered by bolus injection, continuousinfusion, sustained release from implants, or other suitable technique.Typically, a soluble type II IL-1R therapeutic agent will beadministered in the form of a composition comprising purified protein inconjunction with physiologically acceptable carriers, excipients ordiluents. Such carriers will be nontoxic to recipients at the dosagesand concentrations employed. Ordinarily, the preparation of suchcompositions entails combining the type II IL-1R with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with conspecific serum albumin are exemplaryappropriate diluents. Preferably, product is formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents. Appropriate dosages can be determined in trials; generally,shuIL-1R dosages of from about 1 ng/kg/day to about 10 mg/kg/day, andmore preferably from about 500 μg/kg/day to about 5 mg/kg/day, areexpected to induce a biological effect.

Because IL-1R-I and type II IL-1R proteins both bind to IL-1, solubletype II IL-1R proteins are expected to have similar, if not identical,therapeutic activities. For example, soluble human type II IL-1R can beadministered, for example, for the purpose of suppressing immuneresponses in a human. A variety of diseases or conditions are caused byan immune response to alloantigen, including allograft rejection andgraft-versus-host reaction. In alloantigen-induced immune responses,shuIL-1R suppresses lymphoproliferation and inflammation which resultupon activation of T cells. shuIl-1R can therefore be used toeffectively suppress alloantigen-induced immune responses in theclinical treatment of, for example, rejection of allografts (such asskin, kidney, and heart transplants), and graft-versus-host reactions inpatients who have received bone marrow transplants.

Soluble human type II IL-1R can also be used in clinical treatment ofautoimmune dysfunctions, such as rheumatoid arthritis, diabetes andmultiple sclerosis, which are dependent upon the activation of T cellsagainst antigens not recognized as being indigenous to the host.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Isolation of cDNA Encoding Human Type II IL-1R byDirect Expression of Active Protein in CV-1/EBNA-1 Cells

A. Radiolabeling of rIL-1β. Recombinant human IL-1 β was prepared byexpression in E. coli and purification to homogeneity as described byKronheim et al. (Bio/Technology 4:1078, 1986). The IL-1β was labeledwith di-iodo (¹²⁵ I) Bolton-Hunter reagent (New England Nuclear,Glenolden, Pa.). Ten micrograms (0.57 nmol) of protein in 10 uL ofphosphate (0.015 mol/L)-buffered saline (PBS; 0.15 mol/L), pH 7.2, wasmixed with 10 uL of sodium borate (0.1 mol/L)-buffered saline (0.15mol/L), pH 8.5, and reacted with 1 mCi (0.23 nmol) of Bolton-Hunterreagent according to the manufacturer's instructions for 12 hours at 8°C. Subsequently, 30 uL of 2% gelatin and 5 uL of 1 mol/L glycine ethylester were added, and the protein was separated from unreactedBolton-Hunter reagent on a 1 mL bed volume Biogel™ P6 column (Bio-RadLaboratoreis, Richmond, Calif.). Routinely, 50% to 60% incorporation oflabel was observed. Radioiodination yielded specific activities in therange of 1×10¹⁵ to 5×10¹⁵ cpm/mmol-1 (0.4 to 2 atoms I per moleculeprotein), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) revealed a single labeled polypeptide of 17.5 kD, consistantwith previously reported values for IL-1. The labeled protein wasgreater than 98% TCA precipitable, indicating that the ¹²⁵ I wascovalently bound to protein.

B. Construction and Screening of CB23 cDNA library. A CB23 library wasconstructed and screened by direct expression of pooled cDNA clones inthe monkey kidney cell line CV-1/EBNA-1 (which was derived bytransfection of the CV-1 cell line with the gene encoding EBNA-1, asdescribed below) using a mammalian expression vector (pDC406) thatincludes regulatory sequences from SV40, human immunodeficiency virus(HIV), and Epstein-Barr virus (EBV). The CV-1/EBNA-1 cell lineconstitutively expresses EBV nuclear antigen-1 driven from the humancytomegalovirus (CMV) immediate-early enhancer/promoter and thereforeallows the episomal replication of expression vectors such as pDC406that contain the EBV origin of replication. The expression vector usedwas pDC406, a derivative of HAV-EO, described by Dower et al., J.Immunol. 142:4314, 1989), which is in turn a derivative of pDC201 andallows high level expression in the CV-1/EBNA- 1 cell line. pDC406differs from HAV-EO (Dower et al., supra) by the deletion of the intronpresent in the adenovirus 2 tripartite leader sequence in HAV-EO (seedescription of pDC303 below).

The CB23 cDNA library was constructed by reverse transcription ofpoly(A)⁺ mRNA isolated from total RNA extracted from the human B celllymphoblastoid line CB23 (Benjamin & Dower, Blood 75:2017, 1990)substantially as described by Ausubel et al., eds., Current Protocols inMolecular Biology, Vol. 1, 1987. The CB23 cell line is anEBV-transformed cord blood (CB) lymphocyte cell line, which was derivedby using the methods described by Benjamin et al., Proc. Natl. Acad.Sci. USA 81:3547, 1984. Poly(A)⁺ mRNA was isolated by oligo dT cellulosechromatography and double-stranded cDNA was made substantially asdescribed by Gubler and Hoffman, Gene 25:263, 1983. Briefly, thepoly(A)⁺ mRNA was convened to an RNA-cDNA hybrid with reversetranscriptase using random hexanucleotides as a primer. The RNA-cDNAhybrid was then converted into double-stranded eDNA using RNAase H incombination with DNA polymerase I. The resulting double stranded eDNAwas blunt-ended with T4 DNA polymerase. The following two unkinasedoligonucleotides were annealed and blunt end ligated with DNA ligase tothe ends of the resulting blunt-ended eDNA as described by Haymerle, etal., Nucleaic Acids Research, 14: 8615, 1986. ##STR1## In this case onlythe 24-mer oligo will ligate onto the cDNA. The non-ligated oligos wereremoved by gel filtration chromatography at 68° C., leaving 24nucleotide non-self-complementary overhangs on the cDNA. The sameprocedure was used to convert the 5' ends of SalI-cut mammalianexpression vector pDC406 to 24 nucleotide overhangs complementary tothose added to the cDNA. Optimal proportions of adaptored vector andcDNA were ligated in the presence of T4 polynucleotide kinase. Dialyzedligation mixtures were electroporated into E. coli strain DH5α.Approximately 3.9×10⁶ clones were generated and plated in pools ofapproximately 3,000. A sample of each pool was used to prepare frozenglycerol stocks and a sample was used to obtain a pool of plasmid DNA.

The pooled DNA was then used to transfect a sub-confluent layer ofmonkey CV-1/EBNA-1 cells using DEAE-dextran followed by chloroquinetreatment, similar to that described by Luthman et al., Nucl. Acids Res.11:1295 (1983) and McCutchan et al., J. Natl. Cancer Inst. 41:351(1986). CV-1/EBNA-1 cells were derived as follows. The CV-1/EBNA-1 cellline constitutively expresses EBV nuclear antigen-1 driven from the CMVimmediate-early enhancer/promoter. The African Green Monkey kidney cellline, CV-1 (ATCC CCL 70, was cotransfected with 5 μg of pSV2gpt(Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981) and 25 ug ofpDC303/EBNA-1 using a calcium phosphate coprecipitation technique(Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley,N.Y., 1987). pDC303/EBNA-1 was constructed from pDC302 (Mosley et al.,Cell 59:335, 1989) in two steps. First, the intron present in theadenovirus tripartite leader sequence was deleted by replacing a PvuIIto ScaI fragment spanning the intron with the following syntheticoligonucleotide pair to create plasmid pDC303: ##STR2##

Second, a HindIII-AhaII restriction fragment encoding Epstein-Barr virusnuclear antigen I (EBNA-1), and consisting essentially of EBVcoordinates 107,932 to 109,894 (Baer et al., Nature 310:207, 1984), wasthen inserted into the multiple cloning site of pDC303 to create theplasmid pDC303/EBNA-1. The transfected cells were grown in the presenceof hypoxanthine, aminopterin, thymidine, xanthine, and mycophenolic acidaccording to standard methods (Ausubel et al., supra; Mulligan & Berg,supra) to select for the cells that had stably incorporated thetransfected plasmids. The resulting drug resistant colonies wereisolated and expanded individually into cell lines for analysis. Thecell lines were screened for the expression of functional EBNA-1. Onecell line, clone 68, was found to express EBNA-1 using this assay, andwas designated CV-1/EBNA-1. CV-1/EBNA-1 cells were deposited with theAmerican Type Culture Collection on Jun. 5, 1990, under the conditionsof the Budapest Treaty, and assigned number accession CRL 10478.

In order to transfect the CV-1/EBNA-1 cells with the cDNA library, thecells were maintained in complete medium (Dulbecco's modified Eagle'smedia (DMEM) containing 10% (v/v) fetal calf serum (FCS), 50 U/mlpenicillin, 50 U/ml streptomycin, 2 mM L-glutamine) and were plated at adensity of 2×10⁵ cells/well in either 6 well dishes (Falcon) or singlewell chambered slides (Lab-Tek). Both dishes and slides were pretreatedwith 1 ml human fibronectin (10 ug/ml in PBS) for 30 minutes followed by1 wash with PBS. Media was removed from the adherent cell layer andreplaced with 1.5 ml complete medium containing 66.6 μM chloroquinesulfate. 0.2 mls of DNA solution (2 μg DNA, 0.5 mg/ml DEAE-dextran incomplete medium containing chloroquine) was then added to the cells andincubated for 5 hours. Following the incubation, the media was removedand the cells shocked by addition of complete medium containing 10% DMSOfor 2.5 to 20 minutes followed by replacement of the solution with freshcomplete medium. The cells were grown in culture to permit transientexpression of the inserted sequences. These conditions led to an 80%transfection frequency in surviving CV-1/EBNA-1 cells.

After 48 to 72 hours, transfected monolayers of CV-1/EBNA cells wereassayed for expression of IL-1 binding proteins by bindingradioiodinated IL-1β prepared as described above by slideautoradiography. Transfected CV-1/EBNA-1 cells were washed once withbinding medium (RPMI medium 1640 containing 25 mg/ml bovine serumalbumin (BSA), 2 mg/ml sodium azide, 20 mM HEPES, pH 7.2, and 50 mg/mlnonfat dry milk (NFDM)) and incubated for 2 hours at 4° C. with 1 mlbinding medium+NFDM containing 3×10⁻⁹ M ¹²⁵ I-IL-1β. After incubation,cells in the chambered slides were washed three times with bindingbuffer+NFDM, followed by 2 washes with PBS, pH 7.3, to remove unbound¹²⁵ I-IL-1β. The cells were fixed by incubating for 30 minutes at roomtemperature in 10% glutaraldehyde in PBS, pH 7.3, washed twice in PBS,and air dried. The slides were dipped in Kodak GTNB-2 photographicemulsion (6× dilution in water) and exposed in the dark for 48 hours to7 days at 4° C. in a light proof box. The slides were then developed forapproximately 5 minutes in Kodak D19 developer (40 g/500 ml water),rinsed in water and fixed in Agfa G433C fixer. The slides wereindividually examined with a microscope at 25-40× magnification andpositive cells expressing type II IL-1R were identified by the presenceof autoradiographic silver grains against a light background.

Cells in the 6 well plates were washed once with binding buffer+NFDMfollowed by 3 washings with PBS, pH 7.3, to remove unbound ¹²⁵ I-IL-1β.The bound cells were then trypsinized to remove them from the plate andbound ¹²⁵ I-IL-1β were counted on a beta counter.

Using the slide autoradiography approach, approximately 250,000 cDNAswere screened in pools of approximately 3,000 cDNAs until assay of onetransfectant pool showed multiple cells clearly positive for IL-1βbinding. This pool was then partitioned into pools of 500 and againscreened by slide autoradiography and a positive pool was identified.This pool was further partitioned into pools of 75, plated in 6-wellplates and screened by plate binding assays analyzed by quantitation ofbound ¹²⁵ I-IL-1β. The cells were scraped off the plates and counted todetermine which pool of 75 was positive. Individual colonies from thispool of 75 were screened until a single clone (clone 75) was identifiedwhich directed synthesis of a surface protein with detectable IL-1βbinding activity. This clone was isolated, and its insert was sequencedto determine the sequence of the human type II IL-1R eDNA clone 75. ThepDC406 cloning vector containing the human type II IL-1R eDNA clone 75,designated pHuIL-1R-II 75, was deposited with the American Type CultureCollection, Rockville, Md., USA (ATCC) on Jun. 5, 1990 under accessionnumber 68337. The Sequence Listing setting forth the nucleotide (SEQ IDNo:1) and predicted amino acid sequences of clone 75 (SEQ ID No:1 andSEQ ID NO:2) and associated information appears at the end of thespecification immediately prior to the claims.

Example 2 Construction and Expression of cDNAs Encoding Human SolubleType II IL-1R

A cDNA encoding a soluble human type II IL-1R (having the sequence ofamino acids -13-333 of SEQ ID NO:1 ) was constructed by polymerase chainreaction (PCR) amplification using the full length type II IL-1R cDNAclone 75 (SEQ D NO:1) in the vector pDC406 as a template. The following5' oligonucleotide primer (SEQ D NO:7) and 3' oligonucleofide primer(SEQ D NO:8) were first constructed: ##STR3## The 5' primer correspondsto nucleotides 31-51 from the untranslated region of human type II IL-1Rclone 75 (SEQ ID NO:1) with a 5' add-on of a SalI restriction site; thisnucleotide sequence is capable of annealing to the (-) strandcomplementary to nucleotides 31-51 of human clone 75. The 3' primer iscomplementary to nucleotides 1191-1172 (which includes anti-sensenucleotides encoding 3 amino acids of human type II IL-1R clone 75 (SEQID NO: 1 ) and has a 5' add-on of a NotI restriction site and a stopcodon.

The following PCR reagents were added to a 1.5 ml Eppendorf microfugetube: 10 μl of 10× PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.3 at25° C., 15 mM MgCl₂, and 1 mg/ml gelatin) (Perkin-Elmer Cetus, Norwalk,Conn.), 10 μl of a 2 mM solution containing each dNTP (2 mM dATP, 2 mMdCTP, 2 mM dGTP and 2 mM dTTP), 2.5 units (0.5 μl of standard 5000units/ml solution) of Taq DNA polymerase (Perkin-Elmer Cetus), 50 ng oftemplate DNA and 5 121 of a 20 μM solution of each of the aboveoligonucleotide primers and 74.5 μl water to a final volume of 100 μl.The final mixture was then overlaid with 100 μl parafin oil. PCR wascarried out using a DNA thermal cycler (Ericomp, San Diego, Calif.) byinitially denaturing the template at 94° for 90 seconds, reannealing at55° for 75 seconds and extending the cDNA at 72° for 150 seconds. PCRwas carried out for an additional 20 cycles of amplification using astep program (denaturation at 94°, 25 sec; annealing at 55°, 45 sec;extension at 72°, 150 sec.), followed by a 5 minute extension at 72°.

The sample was removed from the parafin oil and DNA extracted byphenolchloroform extraction and spun column chromatography over G-50(Boehringer Mannheim). A 10μl aliquot of the extracted DNA was separatedby electrophoresis on 1% SeaKem™ agarose (FMC BioProducts, Rockland,Me.) and stained with ethidium bromide to confirm that the DNA fragmentsize was consistent with the predicted product.

20 μl of the PCR-amplified cDNA products were then digested with SalIand NotI restriction enzymes using standard procedures. The SalI/NotIrestriction fragment was then separated on a 1.2% Seaplaque™ low gellingtemperature CLGT) agarose, and the band representing the fragment wasisolated. The fragment was ligated into the pDC406 vector by a standard"in gel" ligation method, and the vector was transfected into CV1-EBNAcells and expressed as described above in Example 1.

Example 3 Isolation of cDNAs Encoding Murine Type H IL-1R

Murine type II IL-1R cDNAs were isolated from a cDNA library made fromthe murine pre-B cell line 70Z/3 (ATCC TIB 158), by cross specieshybridization with a human Type II IL-1R probe. A cDNA library wasconstructed in a λ phage vector using λgt10 arms and packaged in vitro(Gigapack®, Stratagene, San Diego) according to the manufacturer'sinstructions. A double-stranded human Type II IL-1R probe was producedby excising an approximately 1.35 kb SalI restriction fragment of thehuman type II IL-1R clone 75 and 32P-labelling the cDNA using randomprimers (Boehringer-Mannheim). The murine cDNA library was amplifiedonce and a total of 5×10⁵ plaques were screened with the human probe in35% formamide (5×SSC, 42° C.). Several murine type II IL-1R cDNA clones(including clone λ2) were isolated; however, none of the clones appearedto be full-length. Nucleotide sequence information obtained from thepartial clones was used to clone a full-length murine type II IL-1R cDNAas follows.

A full-length cDNA clone encoding murine type II IL-1R was isolated bythe method of Rapid Amplification of cDNA Ends (RACE) described byFrohman et al., Proc. Natl. Acad. Sci. USA 85:8998, 1988, using RNA fromthe murine pre-B cell line 70Z/3. Briefly, the RACE method uses PCR toamplify copies of a region of cDNA between a known point in the cDNAtranscript (determined from nucleotide sequence obtained as describedabove) and the 3' end. An adaptor-primer having a sequence containing 17dT base pairs and an adaptor sequence containing three endonucleaserecognition sites (to place convenient restriction sites at the 3' endof the cDNA) is used to reverse transcribe a population of mRNA andproduce (-) strand cDNA. A primer complementary to a known stretch ofsequence in the 5' untranslated region of the murine type H IL-1R clone2 cDNA, described above, and oriented in the 3' direction is annealedwith the (-) strand cDNA and extended to generate a complementary (+)strand cDNA. The resulting double-strand cDNA is amplified by PCR usingprimers that anneal to the natural 5'-end and synthetic 3'-end poly(A)tail. Details of the RACE procedure are as follows.

The following PCR oligonucleotide primers (d(T)17 adaptor-primer, 5'amplification primer and 3' amplification primer, respectively) werefirst constructed: ##STR4## Briefly, the d(T)₁₇ adapter-primer (SEQ IDNO:9) contains nucleotide sequence anneals to the poly(A)+ region of apopulation mRNA transcripts and is used to generated (-) strand cDNAreverse transcripts from mRNA; it also contains endonuclease restrictionsites for PstI, NotI and BamHI to be introduced into the DNA beingamplified by PCR. The 5' amplification primer (SEQ ID NO:10) correspondsto nucleotides 15-34 from the 5' untranslated region of murine type IIIL-1R clone λ2 with a 5' add-on of a SalI restriction site; thisnucleotide sequence anneals to the (-) strand eDNA generated by reversetranscription with the d(T)₁₇ adaptor-primer and is extended to generate(+) strand eDNA. The 3' primer (SEQ ID NO:11) anneals to the (+) strandDNA having the above endonuclease restriction sites and is extended togenerate a double-stranded full-length cDNA encoding murine type IIIL-1R, which can then be amplified by a standard PCR reaction. Detailsof the PCR procedure are as follows.

Poly(A)⁺ mRNA was isolated by oligo dT cellulose chromatography fromtotal RNA extracted from 70Z/3 cells using standard methods described byManiatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Lab., Cold Spring Harbor, N.Y., 1982) and reverse transcribed asfollows. Approximately 1 μg of poly(A)⁺ mRNA in 16.5 μl of water washeated at 68° C. for 3 minutes and then quenched on ice, and added to 2μl of 10× RTC buffer (500 mM Tris-HCl, pH 8.7 at 22° C., 60 mM MgCl, 400mM KCl, 10 mM DTT, each dNTP at 10 mM), 10 units of RNasin (PromegaBiotech), 0.5 μg of d(T)₁₇ -adapter primer and 10 units of AMV reversetranscriptase (Life Sciences) in a total volume of 20 μl, and incubatedfor a period of 2 hours at 42° C. to reverse transcribe the mRNA andsynthesize a pool of eDNA. The reaction mixture was diluted to 1 ml withTE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) and stored at 4° C.overnight.

Approximately 1 or 5 μl of the cDNA pool was combined with 5 μl of a 20solution of the 5' amplification primer, containing sequencecorresponding to the sequence of nucleotides 15-34 of murine type IIIL-1R clone λ2, 5 μl of a 20 μM solution of the 3' amplification primer,10 μl of 10× PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 8.4, 20° C.),14 mM MgCl₂, and 1 mg/ml gelatin), 4 μl of 5 mM each dNTP (containing 5mM dATP, 5 mM dCTP, 5 mM dGTP and 5 mM dTTP), 2.5 units (0.5 μl ofstandard 5000 units/ml solution) of Taq DNA polymerase (Perkin-ElmerCetus Instruments), diluted to a volume of 100 μl. The final mixture wasthen overlaid with 100 μl parafin oil. PCR was carded out using a DNAthermal cycler (Perkin-Elmer/Cetus) by initially denaturing the templateat 94° for 90 seconds, reannealing at 64° for 75 seconds and extendingthe cDNA at 72° for 150 seconds. PCR was carried out for an additional25 cycles of amplification using the following step program(denaturation at 94° for 25 sec; annealing at 55° for 45 sec; extensionat 72° for 150 sec.), followed by a 7 minute final extension at 72°.

The sample was removed from the parafin oil and DNA extracted byphenolchloroform extraction and spun column chromatography over G-50(Boehringer Mannheim). A 10 μl aliquot of the extracted DNA wasseparated by electrophoresis on 1% SeaKem™ agarose (FMC BioProducts,Rockland, Me.) and stained with ethidium bromide to confirm that the DNAfragment size was consistent with the predicted product. The gel wasthen blotted and probed with a 5' 610 bp EcoRI fragment of murine typeII IL-1R clone λ2 from above to confirm that the band contained DNAencoding murine type II IL-1R.

The PCR-amplified cDNA products were then concentrated by centrifugationin an Eppendorf microfuge at full speed for 20 min., followed by ethanolprecipitation in 1/10 volume sodium acetate (3 M) and 2.5 volumeethanol. 30 μl of the concentrate was digested with SalI and NotIrestriction enzymes using standard procedures. The SalI/NotI restrictionfragment was then separated on a 1.2% LGT agarose gel, and the bandrepresenting the fragment was isolated. The restriction fragments werethen purified from the agarose using GeneClean™ (Bio-101, La Jolla,Calif.).

The resulting purified restriction fragment was ligated into the pDC406vector, which was then transfected into CV1-EBNA cells and expressed asdescribed above in Example 1.

The Sequence Listing setting forth the nucleotide (SEQ D No: 12) andpredicted amino acid sequences (SEQ ID No:12 and SEQ ID No:13) andassociated information appears at the end of the specificationimmediately prior to the claims.

Example 4 Construction and Expression of cDNAs Encoding Murine SolubleType H IL-1R

A cDNA encoding soluble murine type II IL-1R (having the sequence ofamino acids -13-345 of SEQ ID NO: 12) was constructed by PCRamplification 70Z/3 poly(A)⁺ mRNA as a template and the followingprocedure as described for the full length clone encoding murine type IIIL-1R. The following PCR oligonucleotide primers (d(T)₁₇ adaptor-primer,5' amplification primer and Y amplification primer, respectively) wereconstructed: ##STR5## The d(T)₁₇ adaptor-primer and 5' amplificationprimer are identical with SEQ ID NO:9 and SEQ ID NO:10, described inExample 5. The 3' end of SEQ ID NO:12 is complementary to nucleotides1145-1166 of SEQ ID NO:12 and has a 5' add-on of a NotI restriction siteand a stop codon.

A pool of cDNA was synthesized from poly(A)⁺ mRNA using the d(T)₁₇adaptor-primer as described in Example 3. To a 1.5 ml Eppendorfmicrofuge tube was added approximately 1 μl of the eDNA pool, 5 μl of a20 μM solution of the 5' amplification primer, 5 μl of a 20 gM solutionof the 3' amplification primer, 10 gL of 10× PCR buffer (500 mM KCl, 100mM Tris-HCl (pH 8.4 at 20° C.), 14 mM MgCl₂, and 1 mg/ml gelatin), 4 μlof 5 mM each of dNTP (containing 5 mM dATP, 5 mM dCTP, 5 mM dGTP and 5mM dTTP), 2.5 units (0.5 μl of standard 5000 units/ml solution) of TaqDNA polymerase (Perkin-Elmer Cetus Instruments), diluted with 75.4 μlwater to a volume of 100 μl. The final mixture was then overlaid with100 μl parafin oil. PCR was carried out using a DNA thermal cycler(Ericomp) by initially denaturing the template at 94° for 90 seconds,reannealing at 55° for 75 seconds and extending the eDNA at 72° for 150seconds. PCR was carried out for an additional 20 cycles ofamplification using the following step program (denaturation at 94° for25 sec; annealing at 55° for 45 sec; extension at 72° for 150 sec.),followed by a 7 minute final extension at 72°.

The sample was removed from the parafin oil and DNA extracted byphenolchloroform extraction and spun column chromatography over G-50(Boehringer Mannheim). A 10 μl aliquot of the extracted DNA wasseparated by electrophoresis on 1% SeaKem™ agarose (FMC BioProducts,Rockland, Me.) and stained with ethidium bromide to confirm that the DNAfragment size was consistent with the predicted product.

The PCR-amplified eDNA products were then concentrated by centrifugationin an Eppendorf microfuge at full speed for 20 min., followed by ethanolprecipitation in 1/10 volume sodium acetate (3 M) and 2.5 volumeethanol. 50 μl was digested with SalI and NotI restriction enzymes usingstandard procedures. The SalI/NotI restriction fragment was thenseparated on a 1.2% Seaplaque LGT agarose gel, and the band representingthe fragment was isolated. The restriction fragment was then purifiedfrom the isolated band using the following freeze/thaw method. The bandfrom the gel was split into two 175 fragments and placed into two 1.5 mlEppendorf microfuge tubes. 500 μl of isolation buffer (0.15 M NaCl, 10mM Tris, pH 8.0, 1 mM EDTA) was added to each tube and the tubes heatedto 68° C. to melt the gel. The gels were then frozen on dry ice for 10minutes, thawed at room temperature and centrifuged at 4° C. for 30minutes. Supernatants were then removed and placed in a new tube,suspended in 2 mL ethanol, and centrifuged at 4° C. for an additional 30minutes to form a DNA pellet. The DNA pellet was washed with 70%ethanol, centrifuged for 5 minutes, removed from the tube andresuspended in 20 μl TE buffer.

The resulting purified restriction fragments were then ligated into thepDC406 vector. A sample of the ligation was transformed into DHSct andcolonies were analyzed to check for correct plasmids. The vector wasthen transfected into COS-7 cells and expressed as described above inExample 1.

Example 5 Type II IL-1R Binding Studies

The binding inhibition constant of recombinant human type II IL-1R,expressed and purified as described in Example 1 above, was determinedby inhibition binding assays in which varying concentrations of acompetitor (IL-1β or IL-1α) was incubated with a constant amount ofradiolabeled IL-1β or IL-1α and cells expressing the type II IL-1R. Thecompetitor binds to the receptor and prevents the radiolabeled ligandfrom binding to the receptor. Binding assays were performed by aphthalate oil separation method essentially as describe by Dower et al.,J. Immunol. 132:751, 1984 and Park et al., J. Biol. Chem. 261:4177,1986. Briefly, CV1/EBNA cells were incubated in six-well plates (Costar,Cambridge, Mass.) at 4° C. for 2 hours with ¹²⁵ I-IL-1β in 1 ml bindingmedium (Roswell Park Memorial Institute (RPMI) 1640 medium containing 2%BSA, 20 mM Hepes buffer, and 0.2% sodium azide, pH 7.2). Sodium azidewas included to inhibit internalization and degradation of ¹²⁵ I-IL-1 bycells at 37° C. The plates were incubated on a gyratory shaker for 1hour at 37° C. Replicate aliquots of the incubation mixture were thentransferred to polyethylene centfifuge tubes containing a phthalate oilmixture comprising 1.5 parts dibutylphthalate, to 1 partbis(s-ethylhexyl)phthalate. Control tubes containing a 100× molar excessof unlabeled IL- 1β were also included to determine non-specificbinding. The cells with bound ¹²⁵ I-IL-1 were separated from unbound ¹²⁵I-IL-1 by centrifugation for 5 minutes at 15,000×g in an EppendorfMicrofuge. The radioactivity associated with the cells was thendetermined on a gamma counter. This assay (using unlabeled human IL-1βas a competitor to inhibit binding of ¹²⁵ I-IL-1β to type II IL-1R)indicated that the full length human type II IL-1R exhibits biphasicbinding to 1L-1β with a K_(I1) of approximately 19±8×10⁹ and K.sub. 12of approximately 0.2±0.002×10⁹. Using unlabeled human IL-1β to inhibitbinding of ¹²⁵ I-IL-1α to type II IL-1R, the full length human type IIIL-1R exhibited biphasic binding to IL-1β with a K_(I1) of approximately2.0±1×10⁹ and K_(I2) of approximately 0.013±0.003×10⁹.

The binding inhibition constant of the soluble human type II IL-1R,expressed and purified as described in Example 2 above, is determined bya inhibition binding assay in which varying concentrations of an IL-1βcompetitor is incubated with a constant amount of radiolabeled I-IL-1βand CB23 cells (an Epstein Barr virus transformed cord blood Blymphocyte cell line) expressing the type II IL-1R. Binding assays werealso performed by a phtahlate oil separation method essentially asdescribe by Dower et al., J. lmmunol. 132:751, 1984 and Park et al., J.Biol. Chem. 261:4177, 1986. Briefly, COS-7 cells were transfected withthe expression vector pDC406 containing a eDNA encoding the solublehuman type II IL-1R described above. Supernatants from the COS cellswere harvested 3 days after transfection and serially diluted in bindingmedium (Roswell Park Memorial Institute (RPMI) 1640 medium containing 2%BSA, 20 mM Hepes buffer, and 0.2% sodium azide, pH 7.2) in 6 well platesto a volume of 50 μl/well. The supernatants were incubated with 50 μl of9×10⁻¹⁰ M ¹²⁵ I-IL-1β plus 2.5×10⁶ CB23 cells at 8° C. for 2 hours withagitation. Duplicate 60 μl aliquots of the incubation mixture were thentransferred to polyethylene centfifuge tubes containing a phthalate oilmixture comprising 1.5 pans dibutylphthalate, to 1 partbis(s-ethylhexyl)phthalate. A negative control tube containing 3×10⁻⁶ Munlabeled IL-1β was also included to determine non-specific binding(100% inhibition) and a positive control tube containing 50 ml bindingmedium with only radiolabled IL-1β was included to determine maxiumbinding. The cells with bound ¹²⁵ I-IL-1β were separated from unbound¹²⁵ I-IL-1β by centrifugation for 5 minutes at 15,000×g in an EppendorfMicrofuge. Supernatants containing unbound ¹²⁵ I-IL-1β were discardedand the cells were carefully rinsed with ice-cold binding medium. Thecells were then incubated in I ml of trypsin-EDTA at 37° C. for 15minutes and cells were harvested. The radioactivity of the cells wasthen determined on a gamma counter. This inhibition binding assay (usingsoluble human type II IL-1R to inhibit binding of IL-1β) indicated thatthe soluble human type II IL-1R has a K_(I) of approximately 3.5×10⁹M⁻¹. Inhibition of IL-1α binding by soluble human type II IL-1R usingthe same procedure indicated that soluble human type II IL-1R has aK_(I) of 1.4×10⁸ M⁻¹.

Murine type II IL-1R exhibits biphasic binding to IL-1β with a K_(I1) of0.8×10⁹ and a K_(I2) of less then 0.01×10⁹.

Example 6 Type H IL-1R Affinity Crosslinking Studies

Affinity crosslinking studies were performed essentially as described byPark et at., Proc. Natl. Acad. Sci. USA 84:1669, 1987. Recombinant humanIL-1α and IL-1β used in the assays were expressed, purified and labeledas described previously (Dower et al., J. Exp. Med. 162:501, 1985; Doweret al., Nature 324:266, 1986). Recombinant human IL-1 receptorantagonist (IL-1ra) was cloned using the cDNA sequence published byEisenberg et al., Nature 343:341, 1990, expressed by transienttransfection in COS cells, and purified by affinity chromatography on acolumn of soluble human type I IL-1R coupled to affigel, as described byDower et al., J. Immunol. 143:4314, 1989, and eluted at low pH.

Briefly, CV1/EBNA cells (4×10⁷ /ml) expressing recombinant type II IL-1Rwere incubated with ¹²⁵ I-IL-1α or ¹²⁵ I-IL-1β (1 nM) at 4° C. in thepresence and absence of 1 mM excess of unlabeled IL-1 as a specificitycontrol for 2 hours. The cells were then washed andbis(sulfosuccinimidyl)suberate was added to a final concentration of 0.1mg/ml. After 30 min. at 25° C., the cells were washed and resuspended in100 μl of phosphate-buffered saline (PBS)/1% Triton containing 2 mMleupeptin, 2 mM o-phenanthroline, and 2 mM EGTA to prevent proteolysis.Aliquots of the extract supernatants containing equal amounts (CPM) of¹²⁵ I-IL-1 and equal volumes of the specificity controls, were analyzedby SDS/PAGE on a 10% gel using standard techniques.

FIG. 4 shows the results of affinity crosslinking studies conducted asdescribed above, using radiolabeled IL-1α and IL-1β, to compare thesizes of the recombinant murine and human type II IL-1 receptor proteinsto their natural counterparts, and to natural and recombinant murine andhuman type I IL-1 receptors. In general, the sizes of thetransiently-expressed recombinant receptors are similar to the naturalreceptors, although the recombinant proteins migrate slightly faster andas slightly broader bands, possibly as a result of differences inglycosylation patter when over-expressed in CV1/EBNA cells. The resultsalso indicate that the type II IL-1 receptors are smaller than the typeI IL-1 receptors. One particular combination (natural human type Ireceptor with IL-1β) failed to yield specific crosslinking products.Since approximately equal amounts of label were loaded into eachexperimental lane, as indicated by the intensity of the free ligandbands at the bottom of the gels, this combinantion must crosslinkrelatively poorly.

The lane showing natural human type II IL-1 receptor-bearing cellscross-linked with ¹²⁵ I-IL-1α reveals a component in the size range(M_(r) =100,000) of complexes with natural and recombinant type Ireceptors. No such complex can be detected in the lane containingrecombinant type II IL-1 receptor, possibly as a result of low levelexpression of type I IL-1 receptors on the CB23 cells, since these cellscontain trace amounts of type I IL1 receptor mRNA.

Example 7 Preparation of Monoclonal Antibodies to Type H IL-1R

Preparations of purified recombinant type II IL-1R, for example, humantype II IL1R, or transfected COS cells expressing high levels of type IIIL-1R are employed to generate monoclonal antibodies against type IIIL-1R using conventional techniques, for example, those disclosed inU.S. Pat. No. 4,411,993. Such antibodies are likely to be useful ininterfering with IL-1 binding to type II IL-1 R, for example, inameliorating toxic or other undesired effects of IL-1, or as componentsof diagnostic or research assays for IL-1 or soluble type II IL-1R.

To immunize mice, type II IL-1R immunogen is emulsified in completeFreund's adjuvant and injected in amounts ranging from 10-100 μgsubcutaneously and interapefitoneally into Balb/c mice. Ten to twelvedays later, the immunized animals are boosted with additional immunogenemulsified in incomplete Freund's adjuvant and periodically boostedthereafter on a weekly to biweekly immunization schedule. Serum samplesare periodically taken by retro-orbital bleeding or tail-tip excisionfor testing by dot-blot assay (antibody sandwich) or ELISA(enzyme-linked immunosorbent assay), or receptor binding inhibition.Other assay procedures are also suitable. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to the murine myelomacell line NS 1 or Ag8.653. Hybfidoma cell lines generated by thisprocedure are plated in multiple microtiter plates in a HAT selectivemedium (hypoxanthine, aminopterin, and thymidine) to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivitywith type II IL-1R, for example, by adaptations of the techniquesdisclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S. Pat.No. 4,703,004. Positive clones are then injected into the peritonealcavities of syngeneic Balb/c mice to produce ascites containing highconcentrations (>1 mg/ml) of anti-type II IL-1R monoclonal antibody, orgrown in flasks or roller bottles. The resulting monoclonal antibody canbe purified by ammonium sulfate precipitation followed by gel exclusionchromatography, and/or affinity chromatography based on binding ofantibody to Protein A of Staphylococcus aureus or protein G fromStreptococci.

BRIEF DESCRIPTION OF THE SEOUENCE LISTING

SEQ ID NO:1 and SEQ ID NO:2 show the nucleotide sequence and predictedamino acid sequence of human type II IL-1R. The mature peptide encodedby this sequence is defined by amino acids 1-385. The predicted signalpeptide is defined by amino acids -13 through -1. The predictedtransmembrane region is defined by amino acids 331-356.

SEQ ID NO:3-SEQ ID NO:6 are various oligonucleotides used to clone thefull-length human type H IL-1R.

SEQ ID NO:7 and SEQ D NO:8 are oligonucleotide primers used to constructa soluble human type II IL-1R by polymerase chain reaction (PCR).

SEQ ID NO:9-SEQ ID NO: 11 are oligonucleotide primers used to clone afull-length and soluble murine type II IL-1Rs.

SEQ ID NO:12 and SEQ ID NO: 13 show the nucleotide sequence andpredicted amino acid sequence of the full-length murine type II IL-1R.The mature peptide encoded by this sequence is defined by amino acids1-397. The predicted signal peptide is defined by amino acids -13through -1. The predicted transmembrane region is defined by amino acids343-368.

SEQ ID NO: 14 is an oligonucleotide primer used to construct a solublemurine type II IL-1R.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 14                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1357 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: CDNA to MRNA                                              (iii) HYPOTHETICAL: N                                                          (iv) ANTI- SENSE: N                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (G) CELL TYPE: Human B cell lymphoblastoid                                    (H) CELL LINE: CB23                                                           (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: CB23 CDNA                                                        (B) CLONE: pHuIL-lRII75                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 154..1350                                                       (D) OTHER INFORMATION:                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 193..1347                                                       (D) OTHER INFORMATION:                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 154..192                                                        (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CTGGAAAATACATTCTGCTACTCTTAAAAACTAGTGACGC TCATACAAATCAACAGAAAG60               AGCTTCTGAAGGAAGACTTTAAAGCTGCTTCTGCCACGTGCTGCTGGGTCTCAGTCCTCC120               ACTTCCCGTGTCCTCTGGAAGTTGTCAGGAGCAATGTTGCGCTTGTACGTGTTG174                      MetLeuArgLeuTyrValLeu                                                        -13-10                                                                        GTAATGGGAGTTTCTGCCTTCACCCTTCAGCCTGCGGCACACACAGGG222                           ValMetGlyValS erAlaPheThrLeuGlnProAlaAlaHisThrGly                             -51510                                                                        GCTGCCAGAAGCTGCCGGTTTCGTGGGAGGCATTACAAGCGGGAGTTC270                           AlaAla ArgSerCysArgPheArgGlyArgHisTyrLysArgGluPhe                             152025                                                                        AGGCTGGAAGGGGAGCCTGTAGCCCTGAGGTGCCCCCAGGTGCCCTAC318                           Arg LeuGluGlyGluProValAlaLeuArgCysProGlnValProTyr                             303540                                                                        TGGTTGTGGGCCTCTGTCAGCCCCCGCATCAACCTGACATGGCATAAA366                           Trp LeuTrpAlaSerValSerProArgIleAsnLeuThrTrpHisLys                             455055                                                                        AATGACTCTGCTAGGACGGTCCCAGGAGAAGAAGAGACACGGATGTGG414                           AsnAsp SerAlaArgThrValProGlyGluGluGluThrArgMetTrp                             606570                                                                        GCCCAGGACGGTGCTCTGTGGCTTCTGCCAGCCTTGCAGGAGGACTCT462                           AlaGlnAspGly AlaLeuTrpLeuLeuProAlaLeuGlnGluAspSer                             75808590                                                                      GGCACCTACGTCTGCACTACTAGAAATGCTTCTTACTGTGACAAAATG510                           GlyThr TyrValCysThrThrArgAsnAlaSerTyrCysAspLysMet                             95100105                                                                      TCCATTGAGCTCAGAGTTTTTGAGAATACAGATGCTTTCCTGCCGTTC558                           Ser IleGluLeuArgValPheGluAsnThrAspAlaPheLeuProPhe                             110115120                                                                     ATCTCATACCCGCAAATTTTAACCTTGTCAACCTCTGGGGTATTAGTA606                           Ile SerTyrProGlnIleLeuThrLeuSerThrSerGlyValLeuVal                             125130135                                                                     TGCCCTGACCTGAGTGAATTCACCCGTGACAAAACTGACGTGAAGATT654                           CysPro AspLeuSerGluPheThrArgAspLysThrAspValLysIle                             140145150                                                                     CAATGGTACAAGGATTCTCTTCTTTTGGATAAAGACAATGAGAAATTT702                           GlnTrpTyrLys AspSerLeuLeuLeuAspLysAspAsnGluLysPhe                             155160165170                                                                  CTAAGTGTGAGGGGGACCACTCACTTACTCGTACACGATGTGGCCCTG750                           LeuSer ValArgGlyThrThrHisLeuLeuValHisAspValAlaLeu                             175180185                                                                     GAAGATGCTGGCTATTACCGCTGTGTCCTGACATTTGCCCATGAAGGC798                           Glu AspAlaGlyTyrTyrArgCysValLeuThrPheAlaHisGluGly                             190195200                                                                     CAGCAATACAACATCACTAGGAGTATTGAGCTACGCATCAAGAAAAAA846                           Gln GlnTyrAsnIleThrArgSerIleGluLeuArgIleLysLysLys                             205210215                                                                     AAAGAAGAGACCATTCCTGTGATCATTTCCCCCCTCAAGACCATATCA894                           LysGlu GluThrIleProValIleIleSerProLeuLysThrIleSer                             220225230                                                                     GCTTCTCTGGGGTCAAGACTGACAATCCCGTGTAAGGTGTTTCTGGGA942                           AlaSerLeuGly SerArgLeuThrIleProCysLysValPheLeuGly                             235240245250                                                                  ACCGGCACACCCTTAACCACCATGCTGTGGTGGACGGCCAATGACACC990                           ThrGly ThrProLeuThrThrMetLeuTrpTrpThrAlaAsnAspThr                             255260265                                                                     CACATAGAGAGCGCCTACCCGGGAGGCCGCGTGACCGAGGGGCCACGC1038                          His IleGluSerAlaTyrProGlyGlyArgValThrGluGlyProArg                             270275280                                                                     CAGGAATATTCAGAAAATAATGAGAACTACATTGAAGTGCCATTGATT1086                          Gln GluTyrSerGluAsnAsnGluAsnTyrIleGluValProLeuIle                             285290295                                                                     TTTGATCCTGTCACAAGAGAGGATTTGCACATGGATTTTAAATGTGTT1134                          PheAsp ProValThrArgGluAspLeuHisMetAspPheLysCysVal                             300305310                                                                     GTCCATAATACCCTGAGTTTTCAGACACTACGCACCACAGTCAAGGAA1182                          ValHisAsnThr LeuSerPheGlnThrLeuArgThrThrValLysGlu                             315320325330                                                                  GCCTCCTCCACGTTCTCCTGGGGCATTGTGCTGGCCCCACTTTCACTG1230                          AlaSer SerThrPheSerTrpGlyIleValLeuAlaProLeuSerLeu                             335340345                                                                     GCCTTCTTGGTTTTGGGGGGAATATGGATGCACAGACGGTGCAAACAC1278                          Ala PheLeuValLeuGlyGlyIleTrpMetHisArgArgCysLysHis                             350355360                                                                     AGAACTGGAAAAGCAGATGGTCTGACTGTGCTATGGCCTCATCATCAA1326                          Arg ThrGlyLysAlaAspGlyLeuThrValLeuTrpProHisHisGln                             365370375                                                                     GACTTTCAATCCTATCCCAAGTGAAATAAAT1357                                           AspPhe GlnSerTyrProLys                                                        380385                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 398 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetLeuArgLeuTyrValLeuValMe tGlyValSerAlaPheThrLeu                             -13-10-51                                                                     GlnProAlaAlaHisThrGlyAlaAlaArgSerCysArgPheArgGly                              510 15                                                                        ArgHisTyrLysArgGluPheArgLeuGluGlyGluProValAlaLeu                              20253035                                                                      ArgCysProGlnValProTyrTrpLeuTrpAlaSerValSer ProArg                             404550                                                                        IleAsnLeuThrTrpHisLysAsnAspSerAlaArgThrValProGly                              556065                                                                        Gl uGluGluThrArgMetTrpAlaGlnAspGlyAlaLeuTrpLeuLeu                             707580                                                                        ProAlaLeuGlnGluAspSerGlyThrTyrValCysThrThrArgAsn                              85 9095                                                                       AlaSerTyrCysAspLysMetSerIleGluLeuArgValPheGluAsn                              100105110115                                                                  ThrAspAlaPheLeuProPheIle SerTyrProGlnIleLeuThrLeu                             120125130                                                                     SerThrSerGlyValLeuValCysProAspLeuSerGluPheThrArg                              135140 145                                                                    AspLysThrAspValLysIleGlnTrpTyrLysAspSerLeuLeuLeu                              150155160                                                                     AspLysAspAsnGluLysPheLeuSerValArgGlyThrThr HisLeu                             165170175                                                                     LeuValHisAspValAlaLeuGluAspAlaGlyTyrTyrArgCysVal                              180185190195                                                                  LeuTh rPheAlaHisGluGlyGlnGlnTyrAsnIleThrArgSerIle                             200205210                                                                     GluLeuArgIleLysLysLysLysGluGluThrIleProValIleIle                               215220225                                                                    SerProLeuLysThrIleSerAlaSerLeuGlySerArgLeuThrIle                              230235240                                                                     ProCysLysValPheLeuGlyThr GlyThrProLeuThrThrMetLeu                             245250255                                                                     TrpTrpThrAlaAsnAspThrHisIleGluSerAlaTyrProGlyGly                              260265270 275                                                                 ArgValThrGluGlyProArgGlnGluTyrSerGluAsnAsnGluAsn                              280285290                                                                     TyrIleGluValProLeuIlePheAspProValThrArg GluAspLeu                             295300305                                                                     HisMetAspPheLysCysValValHisAsnThrLeuSerPheGlnThr                              310315320                                                                     LeuAr gThrThrValLysGluAlaSerSerThrPheSerTrpGlyIle                             325330335                                                                     ValLeuAlaProLeuSerLeuAlaPheLeuValLeuGlyGlyIleTrp                              340345 350355                                                                 MetHisArgArgCysLysHisArgThrGlyLysAlaAspGlyLeuThr                              360365370                                                                     ValLeuTrpProHisHisGln AspPheGlnSerTyrProLys                                   375380385                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI- SENSE: N                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TCGACTGGAACGAGACGACCTGCT24                                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        ( C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI- SENSE: Y                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GACCTTGCTCTGCTGGACGA20                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI- SENSE: N                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTGTTGGGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGT46                              (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI- SENSE: Y                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GACAACCCGAGCGCCAACTCCTGTTTGAGAAGCGCCAGAAAG GTCA46                             (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI- SENSE: N                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCGTCGACCT AGTGACGCTCATACAAATC29                                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv ) ANTI- SENSE: N                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GCGCGGCCGCTCAGGAGGAGGCTTCCTTGACTG33                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                            (iii) HYPOTHETICAL: N                                                         (iv) ANTI- SENSE: N                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CTGCAGGCGGCCGCGGATCCTTTTTTTTTTTTTTTTT37                                       (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI- SENSE: N                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCGTCGACGGCAAGAAGCAGCAAGGTAC28                                                (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI- SENSE: N                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CTGCAGGCGGCCGCGGATCC2 0                                                       (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1366 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: CDNA to MRNA                                              (iii) HYPOTHETICAL: N                                                         (iv) ANTI- SENSE: N                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mouse                                                           (H) CELL LINE: 7OZ/3                                                           (vii) IMMEDIATE SOURCE:                                                      (A) LIBRARY: 7OZ/3                                                            (B) CLONE: 12                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 85..1317                                                        (D) OTHER INFORMATION:                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 124..1314                                                       (D) OTHER INFORMATION:                                                        (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                             (B) LOCATION: 85..123                                                         (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GTCGACGGCAAGAAGCAGCAAGGTACAAGAATACACAGCTCCAGGCTCCAAGGGTCCTGT60                GCGCTCAGGAAGTTGGTGCGGACAATGTTCATCTTGCTTGTGTTAG TAACT111                       MetPheIleLeuLeuValLeuValThr                                                   -13-10-5                                                                      GGAGTTTCTGCTTTCACCACTCCAACAGTGGTGCAC ACAGGAAAGGTT159                          GlyValSerAlaPheThrThrProThrValValHisThrGlyLysVal                              1510                                                                          TCTGAATCCCCCATTACATCGGAGAAGCCCACAGTC CATGGAGACAAC207                          SerGluSerProIleThrSerGluLysProThrValHisGlyAspAsn                              152025                                                                        TGTCAGTTTCGTGGCAGAGAGTTCAAATCTGAATTGAGG CTGGAAGGT255                          CysGlnPheArgGlyArgGluPheLysSerGluLeuArgLeuGluGly                              303540                                                                        GAACCTGTGGTTCTGAGGTGCCCCTTGGCACCTCACTCCGACATC TCC303                          GluProValValLeuArgCysProLeuAlaProHisSerAspIleSer                              45505560                                                                      AGCAGTTCCCATAGTTTTCTGACCTGGAGTAAATTGGAC TCTTCTCAG351                          SerSerSerHisSerPheLeuThrTrpSerLysLeuAspSerSerGln                              657075                                                                        CTGATCCCAAGAGATGAGCCAAGGATGTGGGTGAAG GGTAACATACTC399                          LeuIleProArgAspGluProArgMetTrpValLysGlyAsnIleLeu                              808590                                                                        TGGATTCTGCCAGCAGTGCAGCAAGACTCTGGTACC TACATTTGCACA447                          TrpIleLeuProAlaValGlnGlnAspSerGlyThrTyrIleCysThr                              95100105                                                                      TTCAGAAACGCATCCCACTGTGAGCAAATGTCTGTGGAA CTCAAGGTC495                          PheArgAsnAlaSerHisCysGluGlnMetSerValGluLeuLysVal                              110115120                                                                     TTTAAGAATACTGAAGCATCTCTGCCTCATGTCTCCTACTTGCAA ATC543                          PheLysAsnThrGluAlaSerLeuProHisValSerTyrLeuGlnIle                              125130135140                                                                  TCAGCTCTCTCCACCACCGGGTTACTAGTGTGCCCTGAC CTGAAAGAA591                          SerAlaLeuSerThrThrGlyLeuLeuValCysProAspLeuLysGlu                              145150155                                                                     TTCATCTCCAGCAACGCTGATGGAAAGATACAGTGG TATAAGGGCGCC639                          PheIleSerSerAsnAlaAspGlyLysIleGlnTrpTyrLysGlyAla                              160165170                                                                     ATACTCTTGGATAAAGGCAATAAGGAATTTCTGAGT GCAGGAGACCCC687                          IleLeuLeuAspLysGlyAsnLysGluPheLeuSerAlaGlyAspPro                              175180185                                                                     ACACGCCTATTGATATCCAACACGTCCATGGACGATGCA GGCTATTAC735                          ThrArgLeuLeuIleSerAsnThrSerMetAspAspAlaGlyTyrTyr                              190195200                                                                     AGATGTGTTATGACATTTACCTACAATGGCCAGGAATACAACATC ACT783                          ArgCysValMetThrPheThrTyrAsnGlyGlnGluTyrAsnIleThr                              205210215220                                                                  AGGAATATTGAACTCCGGGTCAAAGGAGCAACCACGGAA CCCATCCCT831                          ArgAsnIleGluLeuArgValLysGlyAlaThrThrGluProIlePro                              225230235                                                                     GTGATCATTTCTCCCCTGGAGACAATACCAGCATCA TTGGGGTCAAGA879                          ValIleIleSerProLeuGluThrIleProAlaSerLeuGlySerArg                              240245250                                                                     CTGATAGTCCCGTGCAAAGTGTTTCTGGGAACTGGT ACATCTTCCAAC927                          LeuIleValProCysLysValPheLeuGlyThrGlyThrSerSerAsn                              255260265                                                                     ACCATTGTGTGGTGGTTGGCTAACAGCACGTTTATCTCG GCTGCTTAC975                          ThrIleValTrpTrpLeuAlaAsnSerThrPheIleSerAlaAlaTyr                              270275280                                                                     CCAAGAGGCCGTGTGACCGAGGGGCTACACCACCAGTACTCAGAG AAT1023                         ProArgGlyArgValThrGluGlyLeuHisHisGlnTyrSerGluAsn                              285290295300                                                                  GATGAAAACTATGTGGAAGTGTCGCTGATTTTTGATCCA GTCACAAGG1071                         AspGluAsnTyrValGluValSerLeuIlePheAspProValThrArg                              305310315                                                                     GAGGATCTGCATACAGATTTTAAATGTGTTGCCTCG AATCCACGGAGT1119                         GluAspLeuHisThrAspPheLysCysValAlaSerAsnProArgSer                              320325330                                                                     TCTCAGTCACTCCATACCACAGTCAAAGAAGTCTCT TCCACGTTCTCC1167                         SerGlnSerLeuHisThrThrValLysGluValSerSerThrPheSer                              335340345                                                                     TGGAGCATTGCGCTGGCACCTCTGTCTCTGATCATCTTG GTTGTGGGG1215                         TrpSerIleAlaLeuAlaProLeuSerLeuIleIleLeuValValGly                              350355360                                                                     GCAATATGGATGCGCAGACGGTGTAAACGCAGGGCTGGAAAGACA TAT1263                         AlaIleTrpMetArgArgArgCysLysArgArgAlaGlyLysThrTyr                              365370375380                                                                  GGACTGACCAAGCTACGGACTGACAACCAGGACTTCCCT TCCAGCCCA1311                         GlyLeuThrLysLeuArgThrAspAsnGlnAspPheProSerSerPro                              385390395                                                                     AACTAAATAAAGGAAATGAAATAAAAAAAAAAAAAAAAAAAG GATCCGCGGCCGC1366                  Asn                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 410 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      MetPheIleLeuLeuValLeuValThrGlyValSerAla PheThrThr                             -13-10-51                                                                     ProThrValValHisThrGlyLysValSerGluSerProIleThrSer                              51015                                                                         GluLys ProThrValHisGlyAspAsnCysGlnPheArgGlyArgGlu                             20253035                                                                      PheLysSerGluLeuArgLeuGluGlyGluProValValLeuArgCys                               404550                                                                       ProLeuAlaProHisSerAspIleSerSerSerSerHisSerPheLeu                              556065                                                                        ThrTrpSerLysLeu AspSerSerGlnLeuIleProArgAspGluPro                             707580                                                                        ArgMetTrpValLysGlyAsnIleLeuTrpIleLeuProAlaValGln                              8590 95                                                                       GlnAspSerGlyThrTyrIleCysThrPheArgAsnAlaSerHisCys                              100105110115                                                                  GluGlnMetSerValGluLeuLysValPheLysAsnT hrGluAlaSer                             120125130                                                                     LeuProHisValSerTyrLeuGlnIleSerAlaLeuSerThrThrGly                              13514014 5                                                                    LeuLeuValCysProAspLeuLysGluPheIleSerSerAsnAlaAsp                              150155160                                                                     GlyLysIleGlnTrpTyrLysGlyAlaIleLeuLeuAspLysGlyAsn                              165 170175                                                                    LysGluPheLeuSerAlaGlyAspProThrArgLeuLeuIleSerAsn                              180185190195                                                                  ThrSerMetAspAspAla GlyTyrTyrArgCysValMetThrPheThr                             200205210                                                                     TyrAsnGlyGlnGluTyrAsnIleThrArgAsnIleGluLeuArgVal                              215 220225                                                                    LysGlyAlaThrThrGluProIleProValIleIleSerProLeuGlu                              230235240                                                                     ThrIleProAlaSerLeuGlySerArgLeuIleValP roCysLysVal                             245250255                                                                     PheLeuGlyThrGlyThrSerSerAsnThrIleValTrpTrpLeuAla                              260265270275                                                                   AsnSerThrPheIleSerAlaAlaTyrProArgGlyArgValThrGlu                             280285290                                                                     GlyLeuHisHisGlnTyrSerGluAsnAspGluAsnTyrValGluVal                               295300305                                                                    SerLeuIlePheAspProValThrArgGluAspLeuHisThrAspPhe                              310315320                                                                     LysCysValAlaSerAsn ProArgSerSerGlnSerLeuHisThrThr                             325330335                                                                     ValLysGluValSerSerThrPheSerTrpSerIleAlaLeuAlaPro                              3403453 50355                                                                 LeuSerLeuIleIleLeuValValGlyAlaIleTrpMetArgArgArg                              360365370                                                                     CysLysArgArgAlaGlyLysThrTyrGlyLeuT hrLysLeuArgThr                             375380385                                                                     AspAsnGlnAspPheProSerSerProAsn                                                390395                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI- SENSE: N                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GCGCGGCCGCCTAGGAAGAGACTTCTTTGACTGTGG36                                    

We claim:
 1. An isolated and purified biologically active Type II IL-1receptor (Type II IL-1R), selected from the group consisting of:(a) aType II IL-1R having an amino acid sequence as set forth in SEQ ID NO.:2, having an amino terminus at amino acid 1, and a carboxy terminusselected from the group consisting of an amino acid between amino acids330 and 385, inclusive, of SEQ ID NO.:2; (b) a polypeptide having anamino acid sequence as set forth in SEQ ID NO:13, having an aminoterminus at amino acid 1, and a carboxy terminus selected from the groupconsisting of an amino acid between amino acids 342 and 397, inclusive,of SEQ ID NO.:13; (c) a fragment of the polypeptide of (a) or (b), whichfragment is capable of binding IL-1; and (d) a biologically active TypeII IL-1R according to (a), (b) or (c), wherein the Type II IL-1Rpolypeptide contains modifications in the amino acid sequence selectedfrom the group consisting of: inactivated N-linked glycosylation sites;altered KEX2 protease cleavage Sites; deleted or substituted cysteineresidues; conservative amino acid substitutions; and combinationsthereof, which Type II IL-1R binds IL-1.
 2. An isolated and purifiedType II IL-1R according to claim 1, which is a soluble Type II IL-1R. 3.A composition comprising a Type II IL-1R according to claim 1, and asuitable diluent or carrier.
 4. A composition comprising a Type II IL-1Raccording to claim 2, and a suitable diluent or carrier.